Combination therapy for treatment of restenosis

ABSTRACT

Described herein are methods for distributing a combination of a cell division inhibitor (e.g., temsirolimus or paclitaxel) and dexamethasone to a tissue surrounding a blood vessel for treating vascular diseases. Also disclosed are injectable compositions of a cell division inhibitor (e.g., temsirolimus or paclitaxel) and dexamethasone for delivery into the tissue surrounding a blood vessel for treating vascular diseases.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.:17/149,631, filed on Jan. 14, 2021, which is a continuation of U.S.application Ser. No. 16/591,467, filed on Oct. 2, 2019, now U.S. Pat.No. 10,925,863, issued on Feb. 23, 2021, which is a divisional of U.S.application Ser. No. 15/990,167 filed on May 25, 2018, now U.S. Pat. No.10,576,063, issued on Mar. 3, 2020, which claims the benefit of U.S.provisional patent application number 62/511,797, filed on May 26, 2017,and U.S. provisional patent application No. 62/624,528, filed on Jan.31, 2018, which are all hereby incorporated by reference in theirentirety.

BACKGROUND

The present disclosure relates generally to medical methods and devices.More particularly, the present disclosure relates to medical methods andkits for distributing temsirolimus in the tissue surrounding a bloodvessel. Further, the present disclosure relates to medical methods andkits for distributing temsirolimus in combination with a glucocorticoidin the tissue surrounding a blood vessel.

Blockages can form in blood vessels under various disease conditions. Inatherosclerosis, the narrowing of arteries in the body, particularly inthe heart, legs, carotid and renal anatomy, can lead to tissue ischemiafrom lack of blood flow. Mechanical revascularization methods, such asballoon angioplasty, atherectomy, stenting, or surgical endarterectomy,are used to open the blood vessel and to improve blood flow todownstream tissues. Unfortunately, mechanical revascularization can leadto an injury cascade that causes the blood vessel to stiffen and vesselwalls to thicken with a scar-like tissue, which can reduce the bloodflow and necessitate another revascularization procedure. There is agreat desire to reduce the vessel stiffening and thickening followingmechanical revascularization to maintain or improve the patency of theblood vessel.

SUMMARY OF THE INVENTION

The present disclosure provides methods, devices, and compositions fordistributing a combination of a cell division inhibitor and aglucocorticoid into a tissue surrounding a blood vessel for treatingvascular diseases.

Provided herein are pharmaceutical compositions comprising temsirolimusand a glucocorticoid, or their pharmaceutically acceptable saltsthereof. Further provided herein are pharmaceutical compositions whereinthe glucocorticoid is dexamethasone or a pharmaceutically acceptablesalt thereof. Further provided herein are pharmaceutical compositionswherein the ratio (by weight) of temsirolimus to the glucocorticoid, orvice versa, is between 10:1 to 1:1. Further provided herein arepharmaceutical compositions wherein the composition is in an injectabledosage form. Further provided herein are pharmaceutical compositionswherein the composition further comprises at least one pharmaceuticallyacceptable excipient. Further provided herein are pharmaceuticalcompositions wherein the concentration of dexamethasone is 1.0-8.0mg/mL. Further provided herein are pharmaceutical compositions whereinthe concentration of dexamethasone is about 3.2 mg/mL. Further providedherein are pharmaceutical compositions wherein the concentration ofdexamethasone is less than 4.0 mg/mL. Further provided herein arepharmaceutical compositions wherein the concentration of temsirolimus is0.01-2.0 mg/mL. Further provided herein are pharmaceutical compositionswherein the concentration of temsirolimus is 0.05-0.5 mg/mL. Furtherprovided herein are pharmaceutical compositions wherein theconcentration of temsirolimus is about 0.1 mg/mL. Further providedherein are pharmaceutical compositions wherein the concentration oftemsirolimus is about 0.4 mg/mL. Further provided herein arepharmaceutical compositions for use in treating restenosis. Furtherprovided herein are pharmaceutical compositions for use in treatingrestenosis below the knee. Further provided herein are pharmaceuticalcompositions for use in treating restenosis above the knee. Furtherprovided herein are pharmaceutical compositions for use in treatingrestenosis in a below-knee popliteal vessel or tibial vessel.

Provided herein are injectable compositions comprising temsirolimus or apharmaceutically acceptable salt thereof, dexamethasone or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable excipient. Further provided herein are injectablecompositions wherein the composition is suitable for adventitialdelivery. Further provided herein are injectable compositions whereinthe composition is suitable for adventitial delivery to a coronaryvessel. Further provided herein are injectable compositions wherein thecomposition is suitable for adventitial delivery to a coronary artery.Further provided herein are injectable compositions wherein thecomposition is suitable for adventitial delivery below the knee. Furtherprovided herein are injectable compositions wherein the composition issuitable for adventitial delivery above the knee. Further providedherein are injectable compositions wherein the composition is suitablefor adventitial delivery to a below-knee popliteal or tibial vessel.Further provided herein are injectable compositions wherein thecomposition is suitable for adventitial delivery to a femoral vessel.Further provided herein are injectable compositions wherein thetherapeutically effective amount of temsirolimus is about 1 μg to 50 mg.Further provided herein are injectable compositions wherein thetherapeutically effective amount of temsirolimus is about 10 μg to 20mg. Further provided herein are injectable compositions wherein thetherapeutically effective amount of temsirolimus is about 100 μg to 15mg. Further provided herein are injectable compositions wherein thetherapeutically effective amount of temsirolimus is about 100 μg to 5mg. Further provided herein are injectable compositions wherein thetherapeutically effective amount is determined by a longitudinal lengthof a target diseased artery. Further provided herein are injectablecompositions wherein the therapeutically effective amount ofdexamethasone is about 0.8 mg to 8 mg per cm of longitudinal length of adisease site in the blood vessel. Further provided herein are injectablecompositions wherein the therapeutically effective amount ofdexamethasone is about 0.05 mg to 10 mg per cm of longitudinal length ofa disease site in the blood vessel and the therapeutically effectiveamount of temsirolimus is about 0.005 mg to 5 mg per cm of longitudinallength of a disease site in the blood vessel. Further provided hereinare injectable compositions wherein the therapeutically effective amountof dexamethasone is about 0.1 mg to 2 mg per cm of longitudinal lengthof a disease site in the blood vessel and the therapeutically effectiveamount of temsirolimus is about 0.025 mg to 1 mg per cm of longitudinallength of the disease site in the blood vessel. Further provided hereinare injectable compositions wherein the injection volume of thecomposition is about 0.01 mL to about 50 mL. Further provided herein areinjectable compositions wherein the injection volume of the compositionis about 0.5 mL to about 20 mL. Further provided herein are injectablecompositions wherein the injection concentration of temsirolimus isabout 0.01 mg/mL to about 2.0 mg/mL. Further provided herein areinjectable compositions wherein the injection concentration oftemsirolimus is about 0.1 mg/mL to about 0.5 mg/mL. Further providedherein are injectable compositions wherein the injection concentrationof temsirolimus is about 0.4 mg/mL. Further provided herein areinjectable compositions wherein the pharmaceutically acceptableexcipient is 0.9% sodium chloride injection USP, dehydrated alcohol,dl-alpha tocopherol, anhydrous citric acid, polysorbate 80, polyethyleneglycol 400, propylene glycol, or a combination thereof. Further providedherein are injectable compositions for use in treating restenosis.Further provided herein are injectable compositions for use in treatingrestenosis below the knee. Further provided herein are injectablecompositions for use in treating restenosis above the knee. Furtherprovided herein are injectable compositions for use in treatingrestenosis in a below-knee popliteal vessel or tibial vessel.

Provided herein are methods of treating a vascular disease in a subjectin need thereof, the method comprising administering to the subject atherapeutically effective amount of a pharmaceutical compositiondescribed herein, wherein the composition is administered by directinjection to a disease site. Further provided herein are methods oftreating a vascular disease wherein the composition is injected though acatheter with a needle. Further provided herein are methods of treatinga vascular disease wherein the composition is injected distal orproximal to the disease site. Further provided herein are methods oftreating a vascular disease wherein the composition is injected at leastabout 2 cm away from the disease site. Further provided herein aremethods of treating a vascular disease wherein the composition isinjected at or adjacent to the disease site. Further provided herein aremethods of treating a vascular disease wherein the composition isadministered by injection into the blood vessel. Further provided hereinare methods of treating a vascular disease wherein the composition isinjected into an adventitial tissue surrounding a blood vessel. Furtherprovided herein are methods of treating a vascular disease wherein thecomposition is injected into a perivascular tissue surrounding the bloodvessel. Further provided herein are methods of treating a vasculardisease wherein the blood vessel is an artery. Further provided hereinare methods of treating a vascular disease wherein the blood vessel is avein. Further provided herein are methods of treating a vascular diseasewherein the artery is a coronary artery or a peripheral artery. Furtherprovided herein are methods of treating a vascular disease wherein theartery is selected from the group consisting of renal artery, cerebralartery, pulmonary artery, and artery in the leg. Further provided hereinare methods of treating a vascular disease wherein the artery is belowthe knee. Further provided herein are methods of treating a vasculardisease wherein the blood vessel is below-knee popliteal vessel ortibial vessel. Further provided herein are methods of treating avascular disease wherein the composition is injected into a blood vesselwall. Further provided herein are methods of treating a vascular diseasewherein the composition is injected into a tissue surrounding the bloodvessel wall. Further provided herein are methods of treating a vasculardisease wherein the therapeutically effective amount of temsirolimus isabout 1 μg to 50 mg. Further provided herein are methods of treating avascular disease wherein the therapeutically effective amount oftemsirolimus is about 10 μg to 20 mg. Further provided herein aremethods of treating a vascular disease wherein the therapeuticallyeffective amount of temsirolimus is about 100 μg to 15 mg. Furtherprovided herein are methods of treating a vascular disease wherein thetherapeutically effective amount of temsirolimus is about 100 μg to 5mg. Further provided herein are methods of treating a vascular diseasewherein the therapeutically effective amount of dexamethasone is about0.8 mg to 8 mg per cm of longitudinal length of the disease site in theblood vessel. Further provided herein are methods of treating a vasculardisease wherein the therapeutically effective amount of dexamethasone isabout 0.05 mg to 10 mg per cm of longitudinal length of the disease sitein the blood vessel and the therapeutically effective amount oftemsirolimus is about 0.005 mg to 5 mg per cm of longitudinal length ofthe disease site in the blood vessel. Further provided herein aremethods of treating a vascular disease wherein the therapeuticallyeffective amount of dexamethasone is about 0.1 mg to 2 mg per cm oflongitudinal length of the disease site in the blood vessel and thetherapeutically effective amount of temsirolimus is about 0.025 mg to 1mg per cm of longitudinal length of the disease site in the bloodvessel. Further provided herein are methods of treating a vasculardisease wherein the injection volume of the composition is about 0.01 mLto about 50 mL. Further provided herein are methods of treating avascular disease wherein the injection volume of the composition isabout 0.5 mL to about 20 mL. Further provided herein are methods oftreating a vascular disease wherein the injection concentration oftemsirolimus is about 0.01 mg/mL to about 2.0 mg/mL. Further providedherein are methods of treating a vascular disease wherein the injectionconcentration of temsirolimus is about 0.1 mg/mL to about 0.4 mg/mL.Further provided herein are methods of treating a vascular diseasewherein the injection concentration of temsirolimus is about 0.4 mg/mL.Further provided herein are methods of treating a vascular diseasewherein the injection concentration of temsirolimus is about 0.1 mg/mL.Further provided herein are methods of treating a vascular diseasewherein 12 months after administration of the pharmaceuticalcomposition, vessel cross-sectional area at the disease site hasdecreased no more than 60%, when compared to vessel cross-sectional areaat the disease site at the time of administration. Further providedherein are methods of treating a vascular disease wherein 12 monthsafter administration of the pharmaceutical composition, vesselcross-sectional area at the disease site has decreased no more than 50%,when compared to vessel cross-sectional area at the disease site at thetime of administration. Further provided herein are methods of treatinga vascular disease wherein 12 months after administration of thepharmaceutical composition, vessel cross-sectional area at the diseasesite has decreased no more than 30%, when compared to vesselcross-sectional area at the disease site at the time of administration.Further provided herein are methods of treating a vascular diseasewherein the subject is human. Further provided herein are methods oftreating a vascular disease wherein the vascular disease is angina,myocardial infarction, congestive heart failure, cardiac arrhythmia,peripheral artery disease, claudication, or critical limb ischemia.Further provided herein are methods of treating a vascular diseasewherein the vascular disease is atherosclerosis, bypass graft failure,transplant vasculopathy, vascular restenosis, or in-stent restenosis.Further provided herein are methods of treating a vascular diseasewherein treating using the pharmaceutical composition results in adecrease in apoptosis at the disease site relative to treating using apharmaceutical composition comprising either temsirolimus or aglucocorticoid. Further provided herein are methods of treating avascular disease wherein treating using the pharmaceutical compositionresults in a decrease in necrosis at the disease site relative totreating using a pharmaceutical composition comprising eithertemsirolimus or a glucocorticoid.

Provided herein are methods of treating a vascular disease in anindividual in need thereof, the method comprising administering to thesubject a therapeutically effective amount of a first pharmaceuticalcomposition and a second pharmaceutical composition, wherein the firstpharmaceutical composition comprises temsirolimus, and the secondpharmaceutical composition comprises a glucocorticoid, wherein the firstcomposition and the second composition are each administered byinjection.

Provided herein are methods of treating a peripheral artery disease in ahuman subject in need thereof, the method comprising administering tothe human subject a therapeutically effective amount of a pharmaceuticalcomposition comprising temsirolimus or pharmaceutically acceptable saltthereof, and a glucocorticoid or pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable excipient, wherein thecomposition is administered by direct injection to or near a diseasesite in a wall of a peripheral artery via a laterally extendinginjection needle of a catheter advanced through vasculature of the humansubject. Further provided herein are methods wherein the compositionfurther comprises a contrast medium for visualizing the injection.

Provided herein are injectable compositions comprising temsirolimus anda glucocorticoid, or pharmaceutically acceptable salts thereof, and apharmaceutically acceptable excipient for use in treating a vasculardisease of a human subject, wherein the composition is suitable foradventitial delivery to a peripheral artery, wherein the composition issuitable for direct injection to a vascular disease site in a wall ofthe peripheral artery via a laterally extending needle from a catheteradvanced through vasculature of the human subject. Further providedherein are methods wherein the composition further comprises a contrastmedium for visualizing the injection.

Provided herein are pharmaceutical compositions comprising a celldivision inhibitor and a glucocorticoid, or their pharmaceuticallyacceptable salts thereof. Further provided herein are pharmaceuticalcompositions wherein the glucocorticoid is dexamethasone or apharmaceutically acceptable salt thereof. Further provided herein arepharmaceutical compositions wherein the ratio (by weight) of a celldivision inhibitor to the glucocorticoid, or vice versa, is between 10:1to 1:1. Further provided herein are pharmaceutical compositions whereinthe composition is in an injectable dosage form. Further provided hereinare pharmaceutical compositions wherein the composition furthercomprises at least one pharmaceutically acceptable excipient. Furtherprovided herein are pharmaceutical compositions wherein theconcentration of dexamethasone is 1.0-8.0 mg/mL. Further provided hereinare pharmaceutical compositions wherein the concentration ofdexamethasone is about 3.2 mg/mL. Further provided herein arepharmaceutical compositions wherein the concentration of dexamethasoneis less than 4.0 mg/mL. Further provided herein are pharmaceuticalcompositions wherein the concentration of a cell division inhibitor is0.01-2.0 mg/mL. Further provided herein are pharmaceutical compositionswherein the concentration of a cell division inhibitor is 0.05-0.5mg/mL. Further provided herein are pharmaceutical compositions whereinthe concentration of a cell division inhibitor is about 0.1 mg/mL.Further provided herein are pharmaceutical compositions wherein theconcentration of a cell division inhibitor is about 0.4 mg/mL. Furtherprovided herein are pharmaceutical compositions for use in treatingrestenosis. Further provided herein are pharmaceutical compositions foruse in treating restenosis below the knee. Further provided herein arepharmaceutical compositions for use in treating restenosis above theknee. Further provided herein are pharmaceutical compositions for use intreating restenosis in a below-knee popliteal vessel or tibial vessel.Further provided herein are pharmaceutical compositions wherein the celldivision inhibitor is paclitaxel.

Provided herein are injectable compositions comprising a cell divisioninhibitor or a pharmaceutically acceptable salt thereof, dexamethasoneor a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable excipient. Further provided herein are injectablecompositions wherein the composition is suitable for adventitialdelivery. Further provided herein are injectable compositions whereinthe composition is suitable for adventitial delivery to a coronaryvessel. Further provided herein are injectable compositions wherein thecomposition is suitable for adventitial delivery to a coronary artery.Further provided herein are injectable compositions wherein thecomposition is suitable for adventitial delivery below the knee. Furtherprovided herein are injectable compositions wherein the composition issuitable for adventitial delivery above the knee. Further providedherein are injectable compositions wherein the composition is suitablefor adventitial delivery to a below-knee popliteal or tibial vessel.Further provided herein are injectable compositions wherein thecomposition is suitable for adventitial delivery to a femoral vessel.Further provided herein are injectable compositions wherein thetherapeutically effective amount of a cell division inhibitor is about 1μg to 50 mg. Further provided herein are injectable compositions whereinthe therapeutically effective amount of a cell division inhibitor isabout 10 μg to 20 mg. Further provided herein are injectablecompositions wherein the therapeutically effective amount of a celldivision inhibitor is about 100 μg to 15 mg. Further provided herein areinjectable compositions wherein the therapeutically effective amount ofa cell division inhibitor is about 100 μg to 5 mg. Further providedherein are injectable compositions wherein the therapeutically effectiveamount is determined by a longitudinal length of a target diseasedartery. Further provided herein are injectable compositions wherein thetherapeutically effective amount of dexamethasone is about 0.8 mg to 8mg per cm of longitudinal length of a disease site in the blood vessel.Further provided herein are injectable compositions wherein thetherapeutically effective amount of dexamethasone is about 0.05 mg to 10mg per cm of longitudinal length of a disease site in the blood vesseland the therapeutically effective amount of a cell division inhibitor isabout 0.005 mg to 5 mg per cm of longitudinal length of a disease sitein the blood vessel. Further provided herein are injectable compositionswherein the therapeutically effective amount of dexamethasone is about0.1 mg to 2 mg per cm of longitudinal length of a disease site in theblood vessel and the therapeutically effective amount of a cell divisioninhibitor is about 0.025 mg to 1 mg per cm of longitudinal length of thedisease site in the blood vessel. Further provided herein are injectablecompositions wherein the injection volume of the composition is about0.01 mL to about 50 mL. Further provided herein are injectablecompositions wherein the injection volume of the composition is about0.5 mL to about 20 mL. Further provided herein are injectablecompositions wherein the injection concentration of a cell divisioninhibitor is about 0.01 mg/mL to about 2.0 mg/mL. Further providedherein are injectable compositions wherein the injection concentrationof a cell division inhibitor is about 0.1 mg/mL to about 0.5 mg/mL.Further provided herein are injectable compositions wherein theinjection concentration of a cell division inhibitor is about 0.4 mg/mL.Further provided herein are injectable compositions wherein thepharmaceutically acceptable excipient is 0.9% sodium chloride injectionUSP, dehydrated alcohol, dl-alpha tocopherol, anhydrous citric acid,polysorbate 80, polyethylene glycol 400, propylene glycol, or acombination thereof. Further provided herein are injectable compositionsfor use in treating restenosis. Further provided herein are injectablecompositions for use in treating restenosis below the knee. Furtherprovided herein are injectable compositions for use in treatingrestenosis above the knee. Further provided herein are injectablecompositions for use in treating restenosis in a below-knee poplitealvessel or tibial vessel. Further provided herein are injectablecompositions wherein the cell division inhibitor is paclitaxel.

Provided herein are methods of treating a vascular disease in a subjectin need thereof, the method comprising administering to the subject atherapeutically effective amount of a pharmaceutical compositiondescribed herein, wherein the composition is administered by directinjection to a disease site. Further provided herein are methods oftreating a vascular disease wherein the composition is injected though acatheter with a needle. Further provided herein are methods of treatinga vascular disease wherein the composition is injected distal orproximal to the disease site. Further provided herein are methods oftreating a vascular disease wherein the composition is injected at leastabout 2 cm away from the disease site. Further provided herein aremethods of treating a vascular disease wherein the composition isinjected at or adjacent to the disease site. Further provided herein aremethods of treating a vascular disease wherein the composition isadministered by injection into a blood vessel. Further provided hereinare methods of treating a vascular disease wherein the composition isinjected into an adventitial tissue surrounding a blood vessel. Furtherprovided herein are methods of treating a vascular disease wherein thecomposition is injected into a perivascular tissue surrounding the bloodvessel. Further provided herein are methods of treating a vasculardisease wherein the blood vessel is an artery. Further provided hereinare methods of treating a vascular disease wherein the blood vessel is avein. Further provided herein are methods of treating a vascular diseasewherein the artery is a coronary artery or a peripheral artery. Furtherprovided herein are methods of treating a vascular disease wherein theartery is selected from the group consisting of renal artery, cerebralartery, pulmonary artery, and artery in the leg. Further provided hereinare methods of treating a vascular disease wherein the artery is belowthe knee. Further provided herein are methods of treating a vasculardisease wherein the blood vessel is below-knee popliteal vessel ortibial vessel. Further provided herein are methods of treating avascular disease wherein the composition is injected into a blood vesselwall. Further provided herein are methods of treating a vascular diseasewherein the composition is injected into a tissue surrounding the bloodvessel wall. Further provided herein are methods of treating a vasculardisease wherein the therapeutically effective amount of a cell divisioninhibitor is about 1 μg to 50 mg. Further provided herein are methods oftreating a vascular disease wherein the therapeutically effective amountof a cell division inhibitor is about 10 μg to 20 mg. Further providedherein are methods of treating a vascular disease wherein thetherapeutically effective amount of a cell division inhibitor is about100 μg to 15 mg. Further provided herein are methods of treating avascular disease wherein the therapeutically effective amount of a celldivision inhibitor is about 100 μg to 5 mg. Further provided herein aremethods of treating a vascular disease wherein the therapeuticallyeffective amount of dexamethasone is about 0.8 mg to 8 mg per cm oflongitudinal length of the disease site in the blood vessel. Furtherprovided herein are methods of treating a vascular disease wherein thetherapeutically effective amount of dexamethasone is about 0.05 mg to 10mg per cm of longitudinal length of the disease site in the blood vesseland the therapeutically effective amount of a cell division inhibitor isabout 0.005 mg to 5 mg per cm of longitudinal length of the disease sitein the blood vessel. Further provided herein are methods of treating avascular disease wherein the therapeutically effective amount ofdexamethasone is about 0.1 mg to 2 mg per cm of longitudinal length ofthe disease site in the blood vessel and the therapeutically effectiveamount of a cell division inhibitor is about 0.025 mg to 1 mg per cm oflongitudinal length of the disease site in the blood vessel. Furtherprovided herein are methods of treating a vascular disease wherein theinjection volume of the composition is about 0.01 mL to about 50 mL.Further provided herein are methods of treating a vascular diseasewherein the injection volume of the composition is about 0.5 mL to about20 mL. Further provided herein are methods of treating a vasculardisease wherein the injection concentration of a cell division inhibitoris about 0.01 mg/mL to about 2.0 mg/mL. Further provided herein aremethods of treating a vascular disease wherein the injectionconcentration of a cell division inhibitor is about 0.1 mg/mL to about0.4 mg/mL. Further provided herein are methods of treating a vasculardisease wherein the injection concentration of a cell division inhibitoris about 0.4 mg/mL. Further provided herein are methods of treating avascular disease wherein the injection concentration of a cell divisioninhibitor is about 0.1 mg/mL. Further provided herein are methods oftreating a vascular disease wherein 12 months after administration ofthe pharmaceutical composition, vessel cross-sectional area at thedisease site has decreased no more than 60%, when compared to vesselcross-sectional area at the disease site at the time of administration.Further provided herein are methods of treating a vascular diseasewherein 12 months after administration of the pharmaceuticalcomposition, vessel cross-sectional area at the disease site hasdecreased no more than 50%, when compared to vessel cross-sectional areaat the disease site at the time of administration. Further providedherein are methods of treating a vascular disease wherein 12 monthsafter administration of the pharmaceutical composition, vesselcross-sectional area at the disease site has decreased no more than 30%,when compared to vessel cross-sectional area at the disease site at thetime of administration. Further provided herein are methods of treatinga vascular disease wherein the subject is human. Further provided hereinare methods of treating a vascular disease wherein the vascular diseaseis angina, myocardial infarction, congestive heart failure, cardiacarrhythmia, peripheral artery disease, claudication, or critical limbischemia. Further provided herein are methods of treating a vasculardisease wherein the vascular disease is atherosclerosis, bypass graftfailure, transplant vasculopathy, vascular restenosis, or in-stentrestenosis. Further provided herein are methods of treating a vasculardisease wherein treating using the pharmaceutical composition results ina decrease in apoptosis at the disease site relative to treating using apharmaceutical composition comprising either a cell division inhibitoror a glucocorticoid. Further provided herein are methods of treating avascular disease wherein treating using the pharmaceutical compositionresults in a decrease in necrosis at the disease site relative totreating using a pharmaceutical composition comprising either a celldivision inhibitor or a glucocorticoid. Further provided herein aremethods wherein the cell division inhibitor is paclitaxel.

Provided herein are methods of treating a vascular disease in anindividual in need thereof, the method comprising administering to thesubject a therapeutically effective amount of a first pharmaceuticalcomposition and a second pharmaceutical composition, wherein the firstpharmaceutical composition comprises a cell division inhibitor, and thesecond pharmaceutical composition comprises a glucocorticoid, whereinthe first composition and the second composition are each administeredby injection. Further provided herein are methods wherein the celldivision inhibitor is paclitaxel.

Provided herein are methods of treating a peripheral artery disease in ahuman subject in need thereof, the method comprising administering tothe human subject a therapeutically effective amount of a pharmaceuticalcomposition comprising a cell division inhibitor or pharmaceuticallyacceptable salt thereof, and a glucocorticoid or pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable excipient,wherein the composition is administered by direct injection to or near adisease site in a wall of a peripheral artery via a laterally extendinginjection needle of a catheter advanced through vasculature of the humansubject. Further provided herein are methods wherein the compositionfurther comprises a contrast medium for visualizing the injection.Further provided herein are methods wherein the cell division inhibitoris paclitaxel.

Provided herein are injectable compositions comprising a cell divisioninhibitor and a glucocorticoid, or pharmaceutically acceptable saltsthereof, and a pharmaceutically acceptable excipient for use in treatinga vascular disease of a human subject, wherein the composition issuitable for adventitial delivery to a peripheral artery, wherein thecomposition is suitable for direct injection to a vascular disease sitein a wall of the peripheral artery via a laterally extending needle froma catheter advanced through vasculature of the human subject. Furtherprovided herein are methods wherein the composition further comprises acontrast medium for visualizing the injection. Further provided hereinare injectable compositions wherein the cell division inhibitor ispaclitaxel.

Incorporation By Reference

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the present disclosure are set forth withparticularity in the appended claims. A better understanding of thefeatures and advantages of the present disclosure will be obtained byreference to the following detailed description that sets forthillustrative embodiments, in which the principles of the presentdisclosure are utilized, and the accompanying drawings of which:

FIG. 1A is a schematic, perspective view of an intraluminal injectioncatheter suitable for use in the methods and systems of the presentdisclosure.

FIG. 1B is a cross-sectional view along line 1B-1B of FIG. 1A.

FIG. 1C is a cross-sectional view along line 1C-1C of FIG. 1A.

FIG. 2A is a schematic, perspective view of the catheter of FIGS. 1A-1Cshown with the injection needle deployed.

FIG. 2B is a cross-sectional view along line 2B-2B of FIG. 2A.

FIG. 3 is a schematic, perspective view of the intraluminal catheter ofFIGS. 1A-1C injecting therapeutic agents into an adventitial spacesurrounding a body lumen in accordance with the methods of the presentdisclosure.

FIG. 4 is a schematic, perspective view of another embodiment of anintraluminal injection catheter useful in the methods of the presentdisclosure.

FIG. 5 is a schematic, perspective view of still another embodiment ofan intraluminal injection catheter useful in the methods of the presentdisclosure, as inserted into one of a patient's body lumens.

FIG. 6 is a perspective view of a needle injection catheter useful inthe methods and systems of the present disclosure.

FIG. 7 is a cross-sectional view of the catheter FIG. 6 shown with theinjection needle in a retracted configuration.

FIG. 8 is a cross-sectional view similar to FIG. 7 , shown with theinjection needle laterally advanced into luminal tissue for the deliveryof therapeutic or diagnostic agents according to the present disclosure.

FIG. 9A is a cross-sectional view of a step in an exemplary fabricationprocess employed to create a free-standing low-modulus patch within ahigher modulus anchor, framework or substrate.

FIG. 9B is a cross-sectional view of a step in an exemplary fabricationprocess employed to create a free-standing low-modulus patch within ahigher modulus anchor, framework or substrate.

FIG. 9C is a cross-sectional view of a step in an exemplary fabricationprocess employed to create a free-standing low-modulus patch within ahigher modulus anchor, framework or substrate.

FIG. 9D is a cross-sectional view of a step in an exemplary fabricationprocess employed to create a free-standing low-modulus patch within ahigher modulus anchor, framework or substrate.

FIG. 9E is a cross-sectional view of a step in an exemplary fabricationprocess employed to create a free-standing low-modulus patch within ahigher modulus anchor, framework or substrate.

FIG. 10A is a cross-sectional view of a step in the inflation process ofan intraluminal injection catheter useful in the methods of the presentdisclosure.

FIG. 10B is a cross-sectional view of a step in the inflation process ofan intraluminal injection catheter useful in the methods of the presentdisclosure.

FIG. 10C is a cross-sectional view of a step in the inflation process ofan intraluminal injection catheter useful in the methods of the presentdisclosure.

FIG. 10D is a cross-sectional view of a step in the inflation process ofan intraluminal injection catheter useful in the methods of the presentdisclosure.

FIG. 11A is a cross-sectional view of the inflated intraluminalinjection catheter useful in the methods of the present disclosure,illustrating the ability to treat multiple lumen diameters.

FIG. 11B is a cross-sectional view of the inflated intraluminalinjection catheter useful in the methods of the present disclosure,illustrating the ability to treat multiple lumen diameters.

FIG. 11C is a cross-sectional view of the inflated intraluminalinjection catheter useful in the methods of the present disclosure,illustrating the ability to treat multiple lumen diameters.

FIG. 12A shows a schematic view of a step in treating a blood vesselaffected by atherosclerosis with delivery of a pharmaceuticalcomposition by injection by a needle through a catheter.

FIG. 12B shows schematic views of a step in treating a blood vesselaffected by atherosclerosis with delivery of a pharmaceuticalcomposition by injection by a needle through a catheter.

FIG. 12C shows schematic views of a step in treating a blood vesselaffected by atherosclerosis with delivery of a pharmaceuticalcomposition by injection by a needle through a catheter.

FIG. 12D shows schematic views of a step in treating a blood vesselaffected by atherosclerosis with delivery of a pharmaceuticalcomposition by injection by a needle through a catheter.

FIG. 12E shows schematic views of a step in treating a blood vesselaffected by atherosclerosis with delivery of a pharmaceuticalcomposition by injection by a needle through a catheter.

FIG. 12F shows schematic views of treating a blood vessel affected byatherosclerosis with delivery of a pharmaceutical composition by a stepin injection by a needle through a catheter.

FIG. 13 shows a flow chart of a method of treating vascular disease in asubject.

FIG. 14A illustrates the percent inhibition of metabolic activity andproliferation in the presence of sirolimus (SIR), dexamethasone (DEX) orsirolimus+dexamethasone (SIR+CONST. DEX) in an experiment measuringmetabolic activity of human aortic vascular smooth muscle cells (VSMCs)in the presence of platelet-derived growth factor (PDGF).

FIG. 14B illustrates the percent inhibition of metabolic activity andproliferation in the presence of temsirolimus (TEM), dexamethasone (DEX)or temsirolimus+dexamethasone (TEM+CONST. DEX) in an experimentmeasuring metabolic activity of human aortic VSMCs in the presence ofplatelet-derived growth factor (PDGF).

FIG. 14C depicts the percent inhibition of metabolic activity andproliferation in the presence of paclitaxel (PACLI), dexamethasone (DEX)or paclitaxel+dexamethasone (PACLI+CONST. DEX) in an experimentmeasuring metabolic activity of human aortic VSMCs in the presence ofplatelet-derived growth factor (PDGF).

FIG. 15A depicts tissue necrosis factor a (TNFα) production in thepresence of increasing concentrations of sirolimus, dexamethasone orsirolimus+50 nM dexamethasone in an experiment measuring TNFα productionof human aortic VSMCs.

FIG. 15B depicts TNFα production in the presence of increasingconcentrations of temsirolimus, dexamethasone or temsirolimus+50 nMdexamethasone in an experiment measuring TNFα production of human aorticVSMCs.

FIG. 15C depicts TNFα production in the presence of increasingconcentrations of paclitaxel, dexamethasone or paclitaxel+50 nMdexamethasone in an experiment measuring TNFα production of human aorticVSMCs.

FIG. 16A depicts interleukin 6 (IL6) production in the presence ofincreasing concentrations of sirolimus, dexamethasone or sirolimus+50 nMdexamethasone in an experiment measuring IL6 cytokine production ofhuman aortic VSMCs.

FIG. 16B depicts IL6 production in the presence of increasingconcentrations of temsirolimus, dexamethasone or temsirolimus+50 nMdexamethasone in an experiment measuring IL6 cytokine production ofhuman aortic VSMCs.

FIG. 16C depicts IL6 production in the presence of increasingconcentrations of paclitaxel or paclitaxel, dexamethasone+50 nMdexamethasone in an experiment measuring IL6 cytokine production ofhuman aortic VSMCs.

FIG. 17A depicts apoptosis by the measurement of Caspase 3 activation inthe presence of increasing concentrations of sirolimus, dexamethasone orsirolimus+50 nM dexamethasone in an experiment measuring apoptosis inhuman aortic VSMCs.

FIG. 17B depicts apoptosis by the measurement of Caspase 3 activation inthe presence of increasing concentrations of temsirolimus, dexamethasoneor temsirolimus+50 nM dexamethasone in an experiment measuring apoptosisin human aortic VSMCs.

FIG. 17C depicts apoptosis by the measurement of Caspase 3 activation inthe presence of increasing concentrations of paclitaxel, dexamethasoneor paclitaxel+50 nM dexamethasone in an experiment measuring apoptosisin human aortic VSMCs.

FIG. 18A depicts necrosis by the measurement of LDH release in thepresence of increasing concentrations of sirolimus, dexamethasone orsirolimus+50 nM dexamethasone in an experiment measuring necrosis inhuman aortic VSMCs.

FIG. 18B depicts necrosis by the measurement of LDH release in thepresence of increasing concentrations of temsirolimus, dexamethasone ortemsirolimus+50 nM dexamethasone in an experiment measuring necrosis inhuman aortic VSMCs.

FIG. 18C depicts necrosis by the measurement of LDH release in thepresence of increasing concentrations of paclitaxel, dexamethasone orpaclitaxel+50 nM dexamethasone in an experiment measuring necrosis inhuman aortic VSMCs.

FIG. 19A depicts inhibition of PDGF-induced VSMC proliferation in thepresence of increasing concentrations of temsirolimus (TEM), paclitaxel(PAC), or dexamethasone (DEX), and then with 500 nanomolar (nM)concentrations of either TEM or PAC as combined with increasingconcentrations of DEX.

FIG. 19B depicts normalized VSMC apoptosis in the presence of PDGF andincreasing concentrations of temsirolimus (TEM), paclitaxel (PAC), ordexamethasone (DEX), and then with 500 nanomolar (nM) concentrations ofeither TEM or PAC as combined with increasing concentrations of DEX.

FIG. 19C depicts normalized VSMC necrosis in the presence of PDGF andincreasing concentrations of temsirolimus (TEM), paclitaxel (PAC), ordexamethasone (DEX), and then with 500 nanomolar (nM) concentrations ofeither TEM or PAC as combined with increasing concentrations of DEX.

FIG. 19D depicts PDGF-induced interleukin-6 (IL6) expression in thepresence of increasing concentrations of temsirolimus (TEM), paclitaxel(PAC), or dexamethasone (DEX), and then with 500 nanomolar (nM)concentrations of either TEM or PAC as combined with increasingconcentrations of DEX.

FIG. 19E depicts PDGF-induced tissue necrosis factor a (TNFα) in thepresence of increasing concentrations of temsirolimus (TEM), paclitaxel(PAC), or dexamethasone (DEX), and then with 500 nanomolar (nM)concentrations of either TEM or PAC as combined with increasingconcentrations of DEX.

FIG. 20 depicts PDGF-induced cell proliferation in the presence ofdecreasing concentrations (100 uM to 1 nM) of temsirolimus (TEM),sirolimus (SIR), paclitaxel (PAC), or dexamethasone (DEX), vehiclecontrol, or cytotoxic control actinomycin D (ACT-D).

DETAILED DESCRIPTION

Provided herein are methods, devices, and compositions for the treatmentof cardiovascular diseases, such as restenosis. Various aspects of themethods, devices, and compositions comprise combinations of an mTORinhibitor (such as temsirolimus) or a cell division inhibitor (such aspaclitaxel) and a glucocorticoid (such as dexamethasone), or theirpharmaceutically acceptable salts. In some cases, the compositions areadministered by direct injection to the disease site.

Diseases and Conditions

Blockages form in blood vessels under various disease conditions.Atherosclerosis, which causes the narrowing, or stenosis, of arteries inthe body, particularly in the heart, legs, carotid, and renal anatomy,leads to tissue ischemia from lack of blood flow. In some instances,atherosclerosis in the coronary arteries causes myocardial infarction,commonly referred to as a heart attack, which can be immediately fatal,or even if survived, causes damage to the heart which can incapacitatethe patient. Other coronary diseases include congestive heart failure,vulnerable or unstable plaque, and cardiac arrhythmias, which causedeath and incapacitation. In addition, peripheral artery disease (PAD),where the arteries in peripheral tissues narrow, most commonly affectsthe leg, renal, and carotid arteries. In some instances, blood clots andthrombus in the peripheral vasculature occlude peripheral blood flow,leading to tissue and organ necrosis. Some patients with PAD experiencecritical limb ischemia that results in ulcers and some instancesrequires amputation in the worst cases. In some instances, PAD in arenal artery causes renovascular hypertension, and clots in the carotidartery embolize and travel to the brain, potentially causing ischemicstroke.

To improve blood flow to downstream tissues, various revascularizationmethods are used to bypass or open the artery. In some instances, arterybypass surgery is an effective treatment for stenosed, or narrowed,arteries resulting from atherosclerosis and other causes, but it is ahighly invasive procedure, expensive, requires substantial hospital andrecovery time. In some instances, mechanical revascularization methodswith balloon angioplasty, atherectomy, stenting, or surgicalendarterectomy are used to open, or dilate, the artery. For example,percutaneous transluminal angioplasty (PTA), commonly referred to asballoon angioplasty, is less invasive, less traumatic, and significantlyless expensive than bypass surgery. In addition, the effectiveness ofballoon angioplasty has improved with the introduction of stenting whichinvolves the placement of a scaffold structure within the artery whichhas been treated by balloon angioplasty. The stent inhibits abruptre-closure of the artery and has some benefit in reducing subsequentrestenosis resulting from hyperplasia. By salvaging blood vessels ratherthan bypassing them, more options are left available to physicians inthe further treatment of the disease.

Unfortunately, in some instances, mechanical revascularizationprocedures lead to an injury cascade that causes the artery to stiffenand arterial walls to thicken with a scar-like tissue, known asneointimal hyperplasia. In some instances, not only does the inner wallof the artery (i.e., the intima) thicken and stiffen in response to theinjury cascade, but the media (i.e., the middle tissue layer of thewall) and the adventitia (i.e., the outer layer of the wall) can thickenand stiffen as well. The thickening (or hyperplasia) and the stiffening(or sclerosis), reduces the blood flow to tissues distal to the affectedsite. As a result, patients who have undergone mechanicalrevascularization procedures in some instances suffer from a highincidence of restenosis resulting from hyperplasia. Restenosis, orrecurrence of stenosis or narrowing, of the blood vessel may necessitateanother revascularization procedure to the affected area again.

Inflammatory restenosis and reocclusion in some cases occurs due tohyperplasia and sclerosis. The resolution of an inflammatory impetus insome instances leads to a stiff, narrow vessel or a functioning arterywith vasomotor actions (including the ability to dilate or constrictbased on signals received from the body's endocrine system. In order tomaintain long-term vascular patency, the vessel must be allowed to healwithout sclerosis or hyperplasia.

Some embodiments herein describe compositions and methods for reducingthe buildup of sclerosis and hyperplasia following mechanicalrevascularization.

In some embodiments, implanting stents coated with anti-proliferativedrugs reduces the occurrence of hyperplasia. For example, mechanicalendovascular revascularization alone leads to patency (the binarymeasure of vessel openness, typically greater than 50% in diametercompared to adjacent non-diseased vessel) rates of 33-55% at one yearand 20-50% at two years, while drug-coated balloons and adventitial drugdelivery have shown an ability to improve patency to better than 80% atone year and 65-70% at 2 years.

Pharmaceutical Compositions Cell Division Inhibitors

Described herein are pharmaceutical compositions comprising a celldivision inhibitor, a glucocorticoid (such as dexamethasone or apharmaceutically acceptable salt thereof), and a pharmaceuticallyacceptable excipient. Some embodiments herein describe a pharmaceuticalcomposition comprising a microtubule stabilizer (such as a taxane or apharmaceutically acceptable salt thereof), a glucocorticoid (such asdexamethasone or a pharmaceutically acceptable salt thereof), and apharmaceutically acceptable excipient. Some embodiments herein describea pharmaceutical composition comprising an mTOR inhibitor (such astemsirolimus or a pharmaceutically acceptable salt thereof), aglucocorticoid (such as dexamethasone or a pharmaceutically acceptablesalt thereof), and a pharmaceutically acceptable excipient. Otherembodiments herein describe a pharmaceutical composition comprising ataxane (such as paclitaxel or a pharmaceutically acceptable saltthereof), a glucocorticoid (such as dexamethasone or a pharmaceuticallyacceptable salt thereof), and a pharmaceutically acceptable excipient.In some embodiments, the pharmaceutical compositions are useful fortreating the diseases and condition described herein (e.g., reducing thebuildup of sclerosis and hyperplasia following mechanicalrevascularization).

mTOR Inhibitors

In certain embodiments, the pharmaceutical compositions described hereincomprise an mTOR inhibitor, such as a -limus drug. In some cases, themTOR inhibitor is sirolimus, everolimus, zotarolimus, deforolimus,biolimus, temsirolimus, or combinations thereof. The pharmaceuticalcompositions disclosed herein in some instances comprise temsirolimus orits pharmaceutically acceptable salts thereof In some embodiments, themTOR inhibitor is Torisel®.

The pharmaceutical compositions disclosed herein in some instancescomprise temsirolimus or its pharmaceutically acceptable salts thereof.In some instances, the pharmaceutical compositions comprisepharmaceutically acceptable excipients. In some instances, thepharmaceutical compositions further comprise excipients including 0.9%sodium chloride injection USP, dehydrated alcohol, dl-alpha tocopherol,anhydrous citric acid, polysorbate 80, polyethylene glycol 400,propylene glycol, or a combination thereof. In some instances, thepharmaceutical compositions comprise nanoparticle formulations. In someinstances, the pharmaceutical compositions further comprise excipientsincluding albumin. In some instances, the pharmaceutical compositionscomprise other excipients commonly used in injectable compositions. Insome instances, the pharmaceutical compositions comprise a contrastagent to aid in visualization of the delivery of the pharmaceuticalcomposition. In some instances, the pharmaceutical compositionscomprising temsirolimus are injectable. In some instances, thepharmaceutical composition is a liquid, a suspension, a solution, or agel.

Taxanes

In certain embodiments, the pharmaceutical compositions described hereincomprise a cell division inhibitor, such as a taxane drug. In somecases, the cell division inhibitor is paclitaxel, docetaxel,cabazitaxel, or combinations thereof. The pharmaceutical compositionsdisclosed herein in some instances comprise paclitaxel or itspharmaceutically acceptable salts thereof In some embodiments, the celldivision inhibitor is Abraxane®.

The pharmaceutical compositions disclosed herein in some instancescomprise paclitaxel or its pharmaceutically acceptable salts thereof Insome instances, the pharmaceutical compositions comprisepharmaceutically acceptable excipients. In some instances, thepharmaceutical compositions further comprise excipients includingcremophor EL, ethanol, or a combination thereof. In some instances, thepharmaceutical compositions comprise nanoparticle formulations. In someinstances, the pharmaceutical compositions further comprise excipientsincluding albumin. In some instances, the pharmaceutical compositionscomprise other excipients commonly used in injectable compositions. Insome instances, the pharmaceutical compositions comprise a contrastagent to aid in visualization of the delivery of the pharmaceuticalcomposition. In some instances, the pharmaceutical compositionscomprising paclitaxel are injectable. In some instances, thepharmaceutical composition is a liquid, a suspension, a solution, or agel. In some instances, pharmaceutical compositions comprise anantihistamine. In some instances, pharmaceutical compositions comprisesolubilizing agents, such as polyols, glycols, or glycerols. In someinstances, pharmaceutical compositions comprise macrogolglycerolricinoleate.

Glucocorticoids

In some instances, the pharmaceutical compositions disclosed hereincomprise one or more glucocorticoids, such as dexamethasone,prenisolone, prednisone, triamcinoclone, hydrocortisone,methylpredinisolone, budesonide, betamethasone, deflazacort,beclomethasone, cortisone, or any other compound that targets theglucocorticoid receptor (GR). In some embodiments, the glucocorticoid isdexamethasone.

The pharmaceutical compositions disclosed herein in some instancescomprise dexamethasone or its pharmaceutically acceptable salts thereof.In some instances, dexamethasone is dexamethasone sodium phosphateinjection. In some instances, the pharmaceutical compositions furthercomprise anhydrous citric acid, sodium sulfite, benzyl alcohol, sodiumcitrate, water for injection, or a combination thereof. In someinstances, the pharmaceutical compositions comprise pharmaceuticallyacceptable excipients. In some instances, the pharmaceuticalcompositions comprise other excipients commonly used in injectablecompositions. In some instances, the pharmaceutical compositionscomprise a contrast agent to aid in visualization of the delivery of thepharmaceutical composition. In some instances, the pharmaceuticalcomposition is injectable. In some instances, the pharmaceuticalcomposition is a liquid, a suspension, a solution, or a gel.

Combination of Cell Division Inhibitors and Glucocorticoids mTORInhibitors and Glucocorticoids

In certain embodiments, a cell division inhibitor, such as an mTORinhibitor and/or a glucocorticoid (such as dexamethasone) as describedherein is administered as a pure chemical. In other embodiments, thecombination of mTOR inhibitor and a glucocorticoid described herein iscombined with a pharmaceutically suitable or acceptable carrier (alsoreferred to herein as a pharmaceutically suitable (or acceptable)excipient, physiologically suitable (or acceptable) excipient, orphysiologically suitable (or acceptable) carrier) selected on the basisof a chosen route of administration and standard pharmaceutical practiceas described, for example, in Remington: The Science and Practice ofPharmacy (Gennaro, 21^(st) Ed. Mack Pub. Co., Easton, PA (2005)). Insome embodiments, an mTOR inhibitor (such as temsirolimus) and aglucocorticoid (e.g., dexamethasone) are each administered as individualcompositions. In some embodiments, individual compositions of an mTORinhibitor or a glucocorticoid are combined with a suitable or acceptableexcipient.

In some embodiments, mTOR inhibitor (such as temsirolimus) and aglucocorticoid (e.g., dexamethasone) are administered as a single,combined composition.

Provided herein are pharmaceutical compositions comprising an mTORinhibitor, and a glucocorticoid together with one or morepharmaceutically acceptable carriers. The carrier(s) (or excipient(s))is acceptable or suitable if the carrier is compatible with the otheringredients of the composition and not deleterious to the recipient(i.e., the subject or patient) of the composition.

In certain embodiments, an mTOR inhibitor and/or a glucocorticoid asdescribed herein is substantially pure, in that it contains less thanabout 5%, or less than about 1%, or less than about 0.1%, of otherorganic small molecules, such as unreacted intermediates or synthesisby-products that are created, for example, in one or more of the stepsof a synthesis method.

In some instances, the pharmaceutical compositions comprise an mTORinhibitor and a glucocorticoid, and one or more pharmaceuticallyacceptable excipients. In some instances, pharmaceutical compositionscomprising an mTOR inhibitor, glucocorticoid, or combination thereofcomprise (by way of non-limiting example) excipients such as 0.9% sodiumchloride injection USP, dehydrated alcohol, dl-alpha tocopherol,anhydrous citric acid, polysorbate 80, polyethylene glycol 400,propylene glycol, benzyl alcohol, sodium citrate, sodium sulfite,albumin, or any combination thereof. In some instances, thepharmaceutical compositions comprise nanoparticles. In some instances,the pharmaceutical compositions comprise other excipients commonly usedin injectable compositions. In some instances, the pharmaceuticalcompositions comprise a contrast agent to aid in visualization of thedelivery of the pharmaceutical composition. In some instances, thepharmaceutical compositions a liquid, a suspension, a solution, or agel. In some instances, the pharmaceutical compositions comprising anmTOR inhibitor, a glucocorticoid, or combination thereof are injectable.In some instances, pharmaceutical compositions comprise excipients thatsolubilize an mTOR inhibitor, a glucocorticoid, or a combinationthereof. In another embodiment, the pharmaceutical compositionscomprising an mTOR inhibitor and a glucocorticoid are provided in adosage form for parenteral administration, which comprises one or morepharmaceutically acceptable excipients or carriers. In some instances,the pharmaceutical compositions comprising an mTOR inhibitor, aglucocorticoid, or combination thereof are injectable. Wherepharmaceutical compositions are formulated for intravenous, cutaneous orsubcutaneous injection, the active ingredient is in the form of aparenterally acceptable aqueous solution, which is pyrogen-free and hasa suitable pH, isotonicity, and stability. Those of relevant skill inthe art are well able to prepare suitable solutions using, for example,isotonic vehicles, such as Sodium Chloride injection, Ringer'sinjection, or Lactated Ringer's injection. In some embodiments,preservatives, stabilizers, excipients, buffers, antioxidants, and/orother additives are included.

In some instances, a pharmaceutical composition comprising an mTORinhibitor (such as temsirolimus) and a glucocorticoid (such asdexamethasone) is administered as a formulation comprising both drugs.In some instances, the percent by weight of temsirolimus to theglucocorticoid or vice versa is between 10:1 to 1:1. In some instances,the percent by weight of temsirolimus to the glucocorticoid or viceversa is between 7:1 to 2:1. In some instances, the percent by weight oftemsirolimus to the glucocorticoid or vice versa is between 5:1 to 1:1.In some instances, the percent by weight of temsirolimus to theglucocorticoid or vice versa is between 3:1 to 1:1 In some instances,the percent by weight of temsirolimus to the glucocorticoid or viceversa is about 10:1, or about 9:1, about 8:1, about 7:1, about 6:1,about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In someinstances, the percent by weight of temsirolimus to the glucocorticoidor vice versa is 10:1 to 1:1, 7:1 to 2:1, 5:1 to 1:1, or 3:1 to 1:1.

Taxanes and Glucocorticoids

In certain embodiments, the combination of a cell division inhibitor(such as paclitaxel) and/or a glucocorticoid (such as dexamethasone) asdescribed herein is administered as a pure chemical. In otherembodiments, the combination of cell division inhibitor and aglucocorticoid described herein is combined with a pharmaceuticallysuitable or acceptable carrier (also referred to herein as apharmaceutically suitable (or acceptable) excipient, physiologicallysuitable (or acceptable) excipient, or physiologically suitable (oracceptable) carrier) selected on the basis of a chosen route ofadministration and standard pharmaceutical practice as described, forexample, in Remington: The Science and Practice of Pharmacy (Gennaro,21^(st) Ed. Mack Pub. Co., Easton, PA (2005)). In some embodiments, acell division inhibitor (such as paclitaxel) and a glucocorticoid (e.g.,dexamethasone) are each administered as individual compositions. In someembodiments, individual compositions of a cell division inhibitor or aglucocorticoid are combined with a suitable or acceptable excipient.

In some embodiments, a cell division inhibitor (such as paclitaxel) anda glucocorticoid (e.g., dexamethasone) are administered as a single,combined composition.

Provided herein are pharmaceutical compositions comprising a celldivision inhibitor, and a glucocorticoid together with one or morepharmaceutically acceptable carriers. The carrier(s) (or excipient(s))is acceptable or suitable if the carrier is compatible with the otheringredients of the composition and not deleterious to the recipient(i.e., the subject or patient) of the composition.

In certain embodiments, a cell division inhibitor and/or aglucocorticoid as described herein is substantially pure, in that itcontains less than about 5%, or less than about 1%, or less than about0.1%, of other organic small molecules, such as unreacted intermediatesor synthesis by-products that are created, for example, in one or moreof the steps of a synthesis method.

In some instances, the pharmaceutical compositions comprise a celldivision inhibitor and a glucocorticoid, and one or morepharmaceutically acceptable excipients. In some instances,pharmaceutical compositions comprising a cell division inhibitor,glucocorticoid, or combination thereof comprise (by way of non-limitingexample) excipients such as 0.9% sodium chloride injection USP,dehydrated alcohol, dl-alpha tocopherol, anhydrous citric acid,polysorbate 80, polyethylene glycol 400, propylene glycol, benzylalcohol, sodium citrate, sodium sulfite, cremophor EL, albumin, or anycombination thereof In some instances, the pharmaceutical compositionscomprise nanoparticles. In some instances, the pharmaceuticalcompositions comprise other excipients commonly used in injectablecompositions. In some instances, the pharmaceutical compositionscomprise a contrast agent to aid in visualization of the delivery of thepharmaceutical composition. In some instances, the pharmaceuticalcompositions comprise a liquid, a suspension, a solution, or a gel. Insome instances, the pharmaceutical compositions comprising a celldivision inhibitor, a glucocorticoid, or combination thereof areinjectable. In some instances, pharmaceutical compositions compriseexcipients that solubilize a cell division inhibitor, a glucocorticoid,or a combination thereof. In another embodiment, the pharmaceuticalcompositions comprising a cell division inhibitor and a glucocorticoidare provided in a dosage form for parenteral administration, whichcomprises one or more pharmaceutically acceptable excipients orcarriers. In some instances, the pharmaceutical compositions comprisinga cell division inhibitor, a glucocorticoid, or combination thereof areinjectable. Where pharmaceutical compositions are formulated forintravenous, cutaneous or subcutaneous injection, the active ingredientis in the form of a parenterally acceptable aqueous solution, which ispyrogen-free and has a suitable pH, isotonicity, and stability. Those ofrelevant skill in the art are well able to prepare suitable solutionsusing, for example, isotonic vehicles, such as Sodium Chlorideinjection, Ringer's injection, or Lactated Ringer's injection. In someembodiments, preservatives, stabilizers, excipients, buffers,antioxidants, and/or other additives are included.

In some instances, a pharmaceutical composition comprising a celldivision inhibitor (such as paclitaxel) and a glucocorticoid (such asdexamethasone) is administered as a formulation comprising both drugs.In some instances, the percent by weight of paclitaxel to theglucocorticoid or vice versa is between 10:1 to 1:1. In some instances,the percent by weight of paclitaxel to the glucocorticoid or vice versais between 7:1 to 2:1. In some instances, the percent by weight ofpaclitaxel to the glucocorticoid or vice versa is between 5:1 to 1:1. Insome instances, the percent by weight of paclitaxel to theglucocorticoid or vice versa is between 3:1 to 1:1 In some instances,the percent by weight of paclitaxel to the glucocorticoid or vice versais about 10:1, or about 9:1, about 8:1, about 7:1, about 6:1, about 5:1,about 4:1, about 3:1, about 2:1, or about 1:1. In some instances, thepercent by weight of paclitaxel to the glucocorticoid or vice versa is10:1 to 1:1, 7:1 to 2:1, 5:1 to 1:1, or 3:1 to 1:1.

Methods of Treatment

In atherosclerosis, bypass graft failure, transplant vasculopathy, andin-stent restenosis, the activation and proliferation of vascular smoothmuscle cells (VSMCs) located in the vessel wall are linked toinflammation, apoptosis, and matrix alterations observed in thesepathophysiologic diseases. An established strategy for the treatment ofthese conditions is to inhibit cellular proliferation through the use ofpaclitaxel. Paclitaxel has cytotoxic effects and may not provide themost durable solution for vascular pathologies. Some embodiments hereindescribe the use of mTOR inhibitors combined with glucocorticoids toprovide a safer and more superior approach to the regulation ofactivated VSMCs compared to anti-proliferatives such as paclitaxel.Other embodiments herein describe the use of cell division inhibitorssuch as paclitaxel combined with glucocorticoids to provide safer andmore superior approaches to the regulation of activated VSMCs comparedto anti-proliferatives such as paclitaxel used alone.

Mechanistic target of rapamycin (mTOR) inhibitors have been identifieddrugs for treating vascular disease. A member of phosphatidylinositol-3kinase-related kinase (PI3K) family, mTOR is involved in regulating cellgrowth, proliferation, cell survival and angiogenesis. In response tophysical insult of revascularization procedure, smooth muscle andendothelial cells in blood vessels activate stress response pathways,which lead to cell proliferation, secretion of pro-inflammatorymediators and extracellular matrix components, and ultimately torestenosis. In some instances, drugs successful in blocking one or moreof the stress response pathways decrease the degree of restenosis. Incertain instances, mTOR inhibitors reduce cellular proliferation andinflammation and have been used successfully in graft-versus-hostdisease, in organ transplant and in some cancers by blocking mTORactivation in response to insulin, growth factors and amino acids.

mTOR inhibitors include the original mTOR inhibitor, sirolimus, alsoknown as rapamycin, and the analogs of sirolimus. These analogs includeeverolimus, zotarolimus, deforolimus, biolimus and temsirolimus.

Temsirolimus is approved for the treatment of renal cell carcinoma(RCC), but it is not approved for the treatment of vascular restenosis.Temsirolimus has been described as a prodrug for sirolimus (i.e.,temsirolimus is metabolized to the active agent sirolimus). As there arefewer metabolic reactions in the vascular tissue compared to the liver(where systemic drugs are generally metabolized), one would not expecttemsirolimus to be efficacious when administered directly to vascularand other luminal walls. Some embodiments provided herein describetemsirolimus as an active agent useful for inhibiting mTOR anddisrupting the mitosis of cells. In some embodiments, temsirolimus isuseful for the treatment of vascular disease. In further or additionalembodiments, temsirolimus is useful for the treatment of vasculardisease via direct injection into vascular or other luminal walls. Infurther or additional embodiments, temsirolimus in combination with aglucocorticoid (e.g., dexamethasone) is useful for the treatment ofvascular disease via direct injection into vascular or other luminalwalls.

Also described herein are methods, devices, and compositions wherein insome embodiments a synergistic effect is observed for the combination of-limus analogs (such as temsirolimus) with corticosteroids (such asdexamethasone) for their ability to reduce the signals causinghyperplasia and sclerosis and to further reduce the cellular activitieswhich apparently cause hyperplasia and sclerosis. Further describedherein are methods wherein, in an attempt to influence the healingprocess, the combination of dexamethasone and temsirolimus, exemplaryagents chosen from the category of corticosteroids (such asglucocorticoids) and mTOR inhibitors, respectively, target the diseaseprocess. In other methods described herein, the combination ofdexamethasone and paclitaxel, exemplary agents chosen from the categoryof corticosteroids (such as glucocorticoids) and cell divisioninhibitors, respectively, target the disease process. In other methodsdescribed herein, the combination of dexamethasone and paclitaxel,exemplary agents chosen from the category of corticosteroids (such asglucocorticoids) and cell division inhibitors, respectively, influencesthe healing process. In some embodiments, the deployment or delivery ofthe drug takes place from the luminal side via drug-coated balloons orpressurized catheters, or from within the vessel wall (the media, theadventitia, or the perivascular tissues) with the use of needleinjection catheters that deploy a needle from the inside to the outsideof the artery. In some embodiments, drug delivery is accomplished usingultrasound guidance and penetration of a needle through the skin andsurrounding tissues to the perivascular area in order to bathe thetarget tissues in the combination drug.

Subjects treated by the methods disclosed herein can exhibit a vasculardisease. In one example, the vascular disease is atherosclerosis in theheart, legs, carotid, or renal blood vessels. In another example, thevascular disease is peripheral artery disease (PAD). In another example,the vascular disease is angina, myocardial infarction, congestive heartfailure, cardiac arrhythmia, peripheral artery disease, claudication, orcritical limb ischemia. In another example, the vascular disease isatherosclerosis, bypass graft failure, transplant vasculopathy, in-stentrestenosis, or restenosis. In one example, the vascular disease is bloodclots, thrombus, or other blockages in a blood vessel that may occludeperipheral blood flow, leading to tissue and organ necrosis. In oneexample, the vascular disease is PAD in renal artery or carotid artery.In some examples, the subject with restenosis had a procedure to improvethe patency of the blood vessel or a revascularization procedurepreviously. In some instances, the vascular disease is restenosis.

In some cases, the disease site of the vascular disease includes bloodvessels and the tissues surrounding the blood vessel. The vasculature ofa subject refers to the circulatory system and in some instancescomprises the arterial system, venous systems, or both arterial andvenous systems and the blood vessels within those systems. In someexamples, the blood vessel is an artery, arteriole, or other bloodvessels of the arterial system. In some examples, the blood vessel is avein, venule, or other blood vessels of the venous system. In oneexample, the artery is a coronary artery or a peripheral artery. In oneexample, the artery is below the knee. In another example, the artery isin the leg above the knee. In another example, the blood vessel isbelow-knee popliteal vessel or tibial vessel. In some examples, theblood vessel is a femoral vessel. In some examples, the artery is renalartery, carotid artery, cerebral artery, pulmonary artery, or artery inthe leg. In some examples, the artery is a femoral artery.

Restenosis is in various tissues and blood vessels in the body. In someinstances, the restenosis is in a coronary vessel. In some instances,the restenosis is in a coronary artery. In some instances, therestenosis is in a peripheral artery. In some instances, the restenosisis in the leg. In other instances, the restenosis is below the knee orin the leg above the knee. In some instances, the restenosis is in abelow-knee popliteal vessel or tibial vessel. In some instances, therestenosis is in a femoral vessel. In other instances, the restenosis isin a femoral artery.

In some instances, the tissue surrounding a blood vessel refers to anytissues outside the endothelial cell wall of the blood vessel that isradially away from the lumen of the blood vessel in a cross section andincludes plaque and calcification. In some instances, the tissuesurrounding a blood vessel comprises adventitial tissue, perivasculartissue, or any tissue surrounding the endothelial wall of a bloodvessel. In some instances, adventitial tissue is also known asadventitia or tunica adventitia or tunica externa. In some instances,adventitial tissue is outside of the external elastic membrane. In someinstances, the tissue surrounding a blood vessel is tissues outside thetunica intima of the blood vessel. In some instances, the tissuesurrounding a blood vessel is tissues outside the tunica media of theblood vessel. In some instances, the tissue surrounding a blood vesselis tissues outside the internal elastic membrane. In some instances, thetissue is a connective tissue. In some instances, the tissue is diseasedtissue such as plaque, fibrosis, calcification, or combinations ofdiseased and healthy tissues.

In some instances, “patency” refers to blood vessel openness. In someinstances, patency at the disease site refers to patency of the bloodvessel, or blood vessel openness, at the disease site. In someinstances, vessel cross-sectional area at the disease site refers topatency of the blood vessel at the disease site. In some instances,vessel cross-sectional area is determined by angiography. In someinstances, the angiography is quantitative vascular angiography (QVA).In other instances, vessel cross-sectional area is determined byintravascular ultrasound (IVUS). In some instances, patency is describedas percent of diameter of the lumen of the blood vessel that is open andunobstructed. In some instances, patency is described as percent ofcross sectional area of the lumen of the blood vessel, or vesselcross-sectional area, that is open and unobstructed. In other instances,patency is the percent of luminal volume that is open and unobstructed.In some instances, patency requires determination of the boundaries ofthe endothelial wall of the blood vessel. In some instances, a bloodvessel that is completely open and unobstructed has 100% patency; i.e.,the blood vessel has a cross-sectional area that is healthy and typicalof a normal, healthy blood vessel in the same part of the body. In someinstances, a blood vessel that is completely blocked and obstructed has0% patency. In some instances, patency is the binary measure of opennessgreater than 50% in diameter compared to adjacent non-diseased vessel.In some instances, patency is the binary measure of openness greaterthan 50% in cross-sectional area compared to adjacent non-diseasedvessel. In some instances, patency is the binary measure of opennessgreater than 50% in luminal volume compared to adjacent non-diseasedvessel.

Dosages/Administration

In some instances, a “therapeutically effective amount” refers to anamount of drug (e.g., temsirolimus or dexamethasone) that increasesvessel cross-sectional area at the disease site. In some instances,therapeutically effective refers to increasing the vesselcross-sectional area at the disease site after administration of apharmaceutical composition. In some instances, therapeutically effectiverefers to minimally decreasing the vessel cross-sectional area at thedisease site after administration when compared to the vesselcross-sectional area at the disease site at the time of administration.In some instances, therapeutically effective refers to increasing thevessel cross-sectional area at the disease site. In some instances,therapeutically effective refers to increasing minimally the vesselcross-sectional area at the disease site after administration whencompared to the vessel cross-sectional area at the disease site at thetime of administration. In some instances, therapeutically effectiverefers to decreasing the vessel cross-sectional area at the disease siteno more than 30%, 20%, 10%, or 0% when compared to the vesselcross-sectional area at the disease site at the time of administration;in other words the patency decreases no more than 30%, 20%, 10%, or 0%when compared to the patency at the disease site at the time ofadministration. In some instances, the vessel cross-sectional area atthe disease site decreases no more than 60%, 50%, 40%, 30%, 20%, or 10%when compared to vessel cross-sectional area at the disease site at thetime of administration. In some instances, the vessel cross-sectionalarea at the disease site increases at least 60%, 50%, 40%, 30%, 20%, or10% when compared to vessel cross-sectional area at the disease site atthe time of administration.

In some instances, the pharmaceutical composition is injected at variouslocations at or near the disease site. In some instances, the diseasesite refers to a blood vessel affected by a vascular disease. In someinstances, the disease site refers to a blood vessel with a partial orcomplete blockage of the lumen. In some instances, the disease siterefers to a blood vessel with a vessel cross-sectional area of less than100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 0% of vesselcross-sectional area of an unobstructed vessel as determined from thevessel wall. In some instances, the pharmaceutical composition isinjected distal or proximal to the disease site. In some instances, thepharmaceutical composition is injected at least about 2 cm away from thedisease site. In some instances, the pharmaceutical composition isinjected at or adjacent to the disease site. In some instances, thepharmaceutical composition is injected into a blood vessel. In someinstances, the pharmaceutical composition is injected into anadventitial tissue surrounding a blood vessel. In some instances, thepharmaceutical composition is injected into a perivascular tissuesurrounding a blood vessel.

Temsirolimus administered as part of a combination therapy with aglucocorticoid in some instances has a range of doses that aretherapeutically effective for treating the vascular disease. In someinstances, the therapeutically effective amount of temsirolimus is about1 μg to 50 mg, about 10 μg to 20 mg, about 25 μg to 10 mg, about 1 μg to2 mg, about 10 μg to 500 μg, about 100 μg to about 2 mg or about 100 μgto 500 μg. In some instances, the therapeutically effective amount oftemsirolimus is about 100 μg to 5 mg. In some instances, thetherapeutically effective amount of temsirolimus is about 100 μg to 15mg. In some instances, the therapeutically effective amount oftemsirolimus is about 10 μg, about 25 μg, about 50 μg, about 100 μg,about 500 μg, about 1.0 mg, about 5.0 mg, about 10.0 mg, or about 15.0mg. In some instances, the therapeutically effective volume oftemsirolimus is about 0.01 ml to about 50 ml, about 0.5 ml to about 20ml, about 0.5 ml to about 25 ml, about 0.5 ml to about 5 ml, or about 1ml to about 5 ml. In some instances, the therapeutically effectiveconcentration of temsirolimus is about 0.1 mg/mL to about 0.4 mg/mL,about 0.1 mg/mL to about 0.5 mg/mL, or about 0.01 mg/mL to about 2.0mg/mL. In some instances, the therapeutically effective concentration oftemsirolimus is about 0.1 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about0.4 mg/mL, about 0.5 mg/mL, about 1.0 mg/ml, about 1.5 mg/ml, about 2.0mg/ml, about 2.5 mg/ml, or 3.0 mg/ml. In some instances, thetherapeutically effective amount of temsirolimus is about 0.005 mg to 5mg per cm of longitudinal length of the disease site in the bloodvessel, about 0.025 mg to 1 mg per cm of longitudinal length of thedisease site in the blood vessel, about 0.05 mg to 2 mg per cm oflongitudinal length of the disease site in the blood vessel, or about0.1 mg to 1 mg per cm of longitudinal length of the disease site in theblood vessel. In some instances, the therapeutically effective amount oftemsirolimus is about 0.025 mg to 1 mg per cm of longitudinal length ofthe blood vessel. The longitudinal length of the disease site in theblood vessel, also known as the longitudinal length of the lesion, isabout 1 cm, 5 cm, 10 cm, 20 cm, 30 cm, 40 cm, or 50 cm.

Paclitaxel administered as part of a combination therapy with aglucocorticoid in some instances has a range of doses that aretherapeutically effective for treating the vascular disease. In someinstances, the therapeutically effective amount of paclitaxel is about 1μg to 50 mg, about 10 μg to 20 mg, about 25 μg to 10 mg, about 1 μg to 2mg, about 10 μg to 500 μg, about 100 μg to about 2 mg or about 100 μg to500 μg. In some instances, the therapeutically effective amount ofpaclitaxel is about 10 μg, about 25 μg, about 50 μg, about 100 μg, about500 μg, about 1.0 mg, about 5.0 mg, about 10.0 mg, or about 15.0 mg. Insome instances, the therapeutically effective volume of paclitaxel isabout 0.01 ml to about 50 ml, about 0.5 ml to about 20 ml, about 0.5 mlto about 25 ml, about 0.5 ml to about 5 ml, or about 1 ml to about 5 ml.In some instances, the therapeutically effective concentration ofpaclitaxel is about 0.1 mg/mL to about 0.4 mg/mL, about 0.1 mg/mL toabout 0.5 mg/mL, or about 0.01 mg/mL to about 2.0 mg/mL. In someinstances, the therapeutically effective concentration of paclitaxel isabout 0.1 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL,about 0.5 mg/mL, about 1.0 mg/ml, about 1.5 mg/ml, about 2.0 mg/ml,about 2.5 mg/ml, or 3.0 mg/ml. In some instances, the therapeuticallyeffective amount of paclitaxel is about 0.005 mg to 5 mg per cm oflongitudinal length of the disease site in the blood vessel, about 0.025mg to 1 mg per cm of longitudinal length of the disease site in theblood vessel, about 0.05 mg to 2 mg per cm of longitudinal length of thedisease site in the blood vessel, or about 0.1 mg to 1 mg per cm oflongitudinal length of the disease site in the blood vessel. In someinstances, the therapeutically effective amount of paclitaxel is about0.025 mg to 1 mg per cm of longitudinal length of the blood vessel. Thelongitudinal length of the disease site in the blood vessel, also knownas the longitudinal length of the lesion, is about 1 cm, 5 cm, 10 cm, 20cm, 30 cm, 40 cm, or 50 cm.

A glucocorticoid administered in combination with temsirolimus comprisesa range of dosages that are therapeutically effective for treating thevascular disease. In some instances, the glucocorticoid isdexamethasone. In some instances, the therapeutically effective amountof dexamethasone is about 1 μg to 50 mg, about 10 μg to 20 mg, about 25μg to 10 mg, about 1 μg to 2 mg, about 10 μg to 500 μg, about 100 μg to50 mg, about 100 μg to 20 mg, about 100 μg to mg, about 100 μg to 1 mg,or about 100 μg to 500 μg. In some instances, the therapeuticallyeffective amount of dexamethasone is about 10 μg, about 25 μg, about 50μg, about 100 μg, about 500 μg, about 1.0 mg, about 5.0 mg, about 10.0mg, about 20.0 mg, about 30.0 mg, about 10 mg, or about 50.0 mg. In someinstances, the therapeutically effective volume of dexamethasone isabout 0.01 mL to about 50 mL, about 0.5 mL to about 20 mL, about 0.5 mLto about 25 mL, about 0.5 mL to about 5 mL, 1 mL to 10 mL, or about 1 mLto about 5 mL. In some instances, the therapeutically effectiveconcentration of dexamethasone is about 0.1 mg/mL to about 0.4 mg/mL,about 0.1 mg/mL to about 0.5 mg/mL, about 0.01 mg/mL to about 2.0 mg/mL,or about 1.0 mg/mL to about 10.0 mg/mL. In some instances, thetherapeutically effective concentration of dexamethasone is about 0.1mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5mg/mL, about 1.0 mg/ml, about 1.5 mg/ml, about 2.0 mg/ml, about 2.5mg/ml, about 3.0 mg/ml, about 4.0 mg/mL, about 6.0 mg/mL, about 8.0mg/mL or about 10.0 mg/mL. In some instances, the therapeuticallyeffective amount of dexamethasone is about 0.05 mg to 10 mg per cm oflongitudinal length of the disease site in the blood vessel, about 0.05mg to 7 mg per cm of longitudinal length of the disease site in theblood vessel, about 0.1 mg to 4 mg per cm of longitudinal length of thedisease site in the blood vessel, or about 0.1 mg to 2 mg per cm oflongitudinal length of the disease site in the blood vessel. In someinstances, the therapeutically effective amount of dexamethasone isabout 0.8 mg to 8 mg per cm of longitudinal length of the disease sitein the blood vessel. In some instances, the therapeutically effectiveamount of dexamethasone is about 0.1 mg to 2 mg per cm of longitudinallength of the disease site in the blood vessel. The longitudinal lengthof the disease site in the blood vessel, also known as the longitudinallength of the lesion, is about 1 cm, 5 cm, 10 cm, 20 cm, 30 cm, 40 cm,or 50 cm.

A glucocorticoid administered in combination with paclitaxel comprises arange of dosages that are therapeutically effective for treating thevascular disease. In some instances, the glucocorticoid isdexamethasone. In some instances, the therapeutically effective amountof dexamethasone is about 1 μg to 50 mg, about 10 μg to 20 mg, about 25μg to 10 mg, about 1 μg to 2 mg, about 10 μg to 500 μg, about 100 μg to50 mg, about 100 μg to 20 mg, about 100 μg to 10 mg, about 100 μg to 1mg, or about 100 μg to 500 μg. In some instances, the therapeuticallyeffective amount of dexamethasone is about 10 μg, about 25 μg, about 50μg, about 100 μg, about 500 μg, about 1.0 mg, about 5.0 mg, about 10.0mg, about 20.0 mg, about 30.0 mg, about mg, or about 50.0 mg. In someinstances, the therapeutically effective volume of dexamethasone isabout 0.01 mL to about 50 mL, about 0.5 mL to about 20 mL, about 0.5 mLto about 25 mL, about 0.5 mL to about 5 mL, 1 mL to 10 mL, or about 1 mLto about 5 mL. In some instances, the therapeutically effectiveconcentration of dexamethasone is about 0.1 mg/mL to about 0.4 mg/mL,about 0.1 mg/mL to about 0.5 mg/mL, about 0.01 mg/mL to about 2.0 mg/mL,or about 1.0 mg/mL to about 10.0 mg/mL. In some instances, thetherapeutically effective concentration of dexamethasone is about 0.1mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5mg/mL, about 1.0 mg/ml, about 1.5 mg/ml, about 2.0 mg/ml, about 2.5mg/ml, about 3.0 mg/ml, about 4.0 mg/mL, about 6.0 mg/mL, about 8.0mg/mL or about 10.0 mg/mL. In some instances, the therapeuticallyeffective amount of dexamethasone is about 0.05 mg to 10 mg per cm oflongitudinal length of the disease site in the blood vessel, about 0.05mg to 7 mg per cm of longitudinal length of the disease site in theblood vessel, about 0.1 mg to 4 mg per cm of longitudinal length of thedisease site in the blood vessel, or about 0.1 mg to 2 mg per cm oflongitudinal length of the disease site in the blood vessel. In someinstances, the therapeutically effective amount of dexamethasone isabout 0.8 mg to 8 mg per cm of longitudinal length of the disease sitein the blood vessel. In some instances, the therapeutically effectiveamount of dexamethasone is about 0.1 mg to 2 mg per cm of longitudinallength of the disease site in the blood vessel. The longitudinal lengthof the disease site in the blood vessel, also known as the longitudinallength of the lesion, is about 1 cm, 5 cm, 10 cm, 20 cm, 30 cm, 40 cm,or 50 cm.

A pharmaceutical composition of temsirolimus administered in combinationwith a glucocorticoid comprises a range of dosages that aretherapeutically effective for treating the vascular disease. In someinstances, the therapeutically effective amount of temsirolimus is about1 μg to 50 mg, about 10 μg to 20 mg, about 25 μg to 10 mg, about 1 μg to2 mg, about 10 μg to 500 μg, about 100 μg to about 2 mg or about 100 μgto 500 μg. In some instances, the therapeutically effective amount oftemsirolimus is about 10 μg, about 25 μg, about 50 μg, about 100 μg,about 500 μg, about 1.0 mg, about 5.0 mg, about 10.0 mg, or about 15.0mg. In some instances, the therapeutically effective volume oftemsirolimus is about 0.01 ml to about 50 ml, about 0.5 ml to about 20ml, about 0.5 ml to about 25 ml, about 0.5 ml to about 5 ml, or about 1ml to about 5 ml. In some instances, the therapeutically effectiveconcentration of temsirolimus is about 0.1 mg/mL to about 0.4 mg/mL,about 0.1 mg/mL to about 0.5 mg/mL, or about 0.01 mg/mL to about 2.0mg/mL. In some instances, the therapeutically effective concentration oftemsirolimus is about 0.1 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about0.4 mg/mL, about 0.5 mg/mL, about 1.0 mg/ml, about 1.5 mg/ml, about 2.0mg/ml, about 2.5 mg/ml, or 3.0 mg/ml. In some instances, thetherapeutically effective amount of temsirolimus is about 0.005 mg to 5mg per cm of longitudinal length of the disease site in the bloodvessel, about 0.025 mg to 1 mg per cm of longitudinal length of thedisease site in the blood vessel, about 0.05 mg to 2 mg per cm oflongitudinal length of the disease site in the blood vessel, or about0.1 mg to 1 mg per cm of longitudinal length of the disease site in theblood vessel. In some instances, the therapeutically effective amount oftemsirolimus is about 0.025 mg to 1 mg per cm of longitudinal length ofthe blood vessel. The longitudinal length of the disease site in theblood vessel, also known as the longitudinal length of the lesion, isabout 1 cm, 5 cm, 10 cm, 20 cm, 30 cm, 40 cm, or 50 cm.

Alternatively, a pharmaceutical composition of paclitaxel administeredin combination with a glucocorticoid comprises a range of dosages thatare therapeutically effective for treating the vascular disease. In someinstances, the therapeutically effective amount of paclitaxel is about 1μg to 50 mg, about 10 μg to 20 mg, about 25 μg to 10 mg, about 1 μg to 2mg, about 10 μg to 500 μg, about 100 μg to about 2 mg or about 100 μg to500 μg. In some instances, the therapeutically effective amount ofpaclitaxel is about 10 μg, about 25 μg, about 50 μg, about 100 μg, about500 μg, about 1.0 mg, about 5.0 mg, about 10.0 mg, or about 15.0 mg. Insome instances, the therapeutically effective volume of paclitaxel isabout 0.01 ml to about 50 ml, about 0.5 ml to about 20 ml, about 0.5 mlto about 25 ml, about 0.5 ml to about 5 ml, or about 1 ml to about 5 ml.In some instances, the therapeutically effective concentration ofpaclitaxel is about 0.1 mg/mL to about 0.4 mg/mL, about 0.1 mg/mL toabout 0.5 mg/mL, or about 0.01 mg/mL to about 2.0 mg/mL. In someinstances, the therapeutically effective concentration of paclitaxel isabout 0.1 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL,about 0.5 mg/mL, about 1.0 mg/ml, about 1.5 mg/ml, about 2.0 mg/ml,about 2.5 mg/ml, or 3.0 mg/ml. In some instances, the therapeuticallyeffective amount of paclitaxel is about 0.005 mg to 5 mg per cm oflongitudinal length of the disease site in the blood vessel, about 0.025mg to 1 mg per cm of longitudinal length of the disease site in theblood vessel, about 0.05 mg to 2 mg per cm of longitudinal length of thedisease site in the blood vessel, or about 0.1 mg to 1 mg per cm oflongitudinal length of the disease site in the blood vessel. In someinstances, the therapeutically effective amount of paclitaxel is about0.025 mg to 1 mg per cm of longitudinal length of the blood vessel. Thelongitudinal length of the disease site in the blood vessel, also knownas the longitudinal length of the lesion, is about 1 cm, 5 cm, 10 cm, 20cm, 30 cm, 40 cm, or 50 cm.

A pharmaceutical composition of a glucocorticoid administered incombination with temsirolimus comprises a range of dosages that aretherapeutically effective for treating the vascular disease. In someinstances, the glucocorticoid is dexamethasone. In some instances, thetherapeutically effective amount of dexamethasone is about 1 μg to 50mg, about 10 μg to 20 mg, about 25 μg to 10 mg, about 1 μg to 2 mg,about 10 μg to 500 μg, about 100 μg to 50 mg, about 100 μg to 20 mg,about 100 μg to 10 mg, about 100 μg to 1 mg, or about 100 μg to 500 μg.In some instances, the therapeutically effective amount of dexamethasoneis about 10 μg, about 25 μg, about 50 μg, about 100 μg, about 500 μg,about 1.0 mg, about 5.0 mg, about 10.0 mg, about 20.0 mg, about 30.0 mg,about 40.0 mg, or about 50.0 mg. In some instances, the therapeuticallyeffective volume of dexamethasone is about 0.01 mL to about 50 mL, about0.5 mL to about 20 mL, about 0.5 mL to about 25 mL, about 0.5 mL toabout 5 mL, 1 mL to 10 mL, or about 1 mL to about 5 mL. In someinstances, the therapeutically effective concentration of dexamethasoneis about 0.1 mg/mL to about 0.4 mg/mL, about 0.1 mg/mL to about 0.5mg/mL, about 0.01 mg/mL to about 2.0 mg/mL, or about 1.0 mg/mL to about10.0 mg/mL. In some instances, the therapeutically effectiveconcentration of dexamethasone is about 0.1 mg/mL, about 0.2 mg/mL,about 0.3 mg/mL, about 0.4 mg/mL, about 0.5 mg/mL, about 1.0 mg/ml,about 1.5 mg/ml, about 2.0 mg/ml, about 2.5 mg/ml, about 3.0 mg/ml,about 4.0 mg/mL, about 6.0 mg/mL, about 8.0 mg/mL or about 10.0 mg/mL.In some instances, the therapeutically effective amount of dexamethasoneis about 0.05 mg to 10 mg per cm of longitudinal length of the diseasesite in the blood vessel, about 0.05 mg to 7 mg per cm of longitudinallength of the disease site in the blood vessel, about 0.1 mg to 4 mg percm of longitudinal length of the disease site in the blood vessel, orabout 0.1 mg to 2 mg per cm of longitudinal length of the disease sitein the blood vessel. In some instances, the therapeutically effectiveamount of dexamethasone is about 0.1 mg to 2 mg per cm of longitudinallength of the disease site in the blood vessel. The longitudinal lengthof the disease site in the blood vessel, also known as the longitudinallength of the lesion, is about 1 cm, 5 cm, 10 cm, 20 cm, 30 cm, 40 cm,or 50 cm.

The dose of the composition comprising temsirolimus and a glucocorticoidas described herein differ, depending upon the patient's condition, thatis, stage of the disease, general health status, age, and other factors.

The dose of the composition comprising paclitaxel and a glucocorticoidas described herein differ, depending upon the patient's condition, thatis, stage of the disease, general health status, age, and other factors.

Pharmaceutical compositions described herein are administered in amanner appropriate to the disease to be treated (or prevented). Anappropriate dose and a suitable duration and frequency of administrationwill be determined by such factors as the condition of the patient, thetype and severity of the patient's disease, the particular form of theactive ingredient, and the method of administration. In general, anappropriate dose and treatment regimen provides the composition(s) in anamount sufficient to provide therapeutic and/or prophylactic benefit(e.g., an improved clinical outcome), or a lessening of symptomseverity. Optimal doses are generally determined using experimentalmodels and/or clinical trials. The optimal dose depends upon the bodymass, weight, or blood volume of the patient.

In one embodiment, the pharmaceutical compositions of temsirolimus and aglucocorticoid described herein, or pharmaceutically acceptable saltsthereof, are used in the preparation of medicaments for the treatment ofdiseases or conditions in a mammal that would benefit fromadministration of any one of the compounds disclosed. In one embodiment,the pharmaceutical compositions of paclitaxel and a glucocorticoiddescribed herein, or pharmaceutically acceptable salts thereof, are usedin the preparation of medicaments for the treatment of diseases orconditions in a mammal that would benefit from administration of any oneof the compounds disclosed. Methods for treating any of the diseases orconditions described herein in a mammal in need of such treatment,involves administration of pharmaceutical compositions that include atleast one compound described herein or a pharmaceutically acceptablesalt, active metabolite, prodrug, or pharmaceutically acceptable solvatethereof, in therapeutically effective amounts to said mammal.

In certain embodiments, the pharmaceutical compositions of temsirolimusand a glucocorticoid described herein are administered for prophylacticand/or therapeutic treatments. In certain embodiments, thepharmaceutical compositions of paclitaxel and a glucocorticoid describedherein are administered for prophylactic and/or therapeutic treatments.In certain therapeutic applications, the compositions are administeredto a patient already suffering from a disease or condition, in an amountsufficient to cure or at least partially arrest at least one of thesymptoms of the disease or condition. Amounts effective for this usedepend on the severity and course of the disease or condition, previoustherapy, the patient's health status, weight, and response to the drugs,and the judgment of the treating physician. Therapeutically effectiveamounts are optionally determined by methods including, but not limitedto, a dose escalation and/or dose ranging clinical trial.

In prophylactic applications, the pharmaceutical composition oftemsirolimus and a glucocorticoid described herein are administered to apatient susceptible to or otherwise at risk of a particular disease,disorder or condition. In prophylactic applications, the pharmaceuticalcomposition of paclitaxel and a glucocorticoid described herein areadministered to a patient susceptible to or otherwise at risk of aparticular disease, disorder or condition. Such an amount is defined tobe a “prophylactically effective amount or dose.” In this use, theprecise amounts also depend on the patient's state of health, weight,and the like. When used in patients, effective amounts for this use willdepend on the severity and course of the disease, disorder or condition,previous therapy, the patient's health status and response to the drugs,and the judgment of the treating physician. In one aspect, prophylactictreatments include administering to a mammal, in which the mammalpreviously experienced at least one symptom of the disease being treatedand is currently in remission, a pharmaceutical composition comprising acompound described herein, or a pharmaceutically acceptable saltthereof, in order to prevent a return of the symptoms of the disease orcondition.

In certain embodiments wherein the patient's condition does not improve,upon the doctor's discretion the administration of a pharmaceuticalcomposition of temsirolimus and a glucocorticoid are administeredchronically, that is, for an extended period of time, includingthroughout the duration of the patient's life in order to ameliorate orotherwise control or limit the symptoms of the patient's disease orcondition. In certain embodiments wherein the patient's condition doesnot improve, upon the doctor's discretion the administration of apharmaceutical composition of paclitaxel and a glucocorticoid areadministered chronically, that is, for an extended period of time,including throughout the duration of the patient's life in order toameliorate or otherwise control or limit the symptoms of the patient'sdisease or condition.

Once improvement of the patient's conditions has occurred, a maintenanceinjection is administered if necessary. Subsequently, in specificembodiments, the dosage or the frequency of administration, or both, isreduced, as a function of the symptoms, to a level at which the improveddisease, disorder or condition is retained. In certain embodiments,however, the patient requires intermittent treatment on a long-termbasis upon any recurrence of symptoms.

Toxicity and therapeutic efficacy of such therapeutic regimens aredetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, including, but not limited to, the determinationof the LD50 and the ED50. The dose ratio between the toxic andtherapeutic effects is the therapeutic index and it is expressed as theratio between LD50 and ED50. In certain embodiments, the data obtainedfrom cell culture assays and animal studies are used in formulating thetherapeutically effective daily dosage range and/or the therapeuticallyeffective unit dosage amount for use in mammals, including humans. Insome embodiments, the dosage amount of the compounds described hereinlies within a range of circulating concentrations that include the ED50with minimal toxicity. In certain embodiments, the dosage range and/orthe unit dosage amount varies within this range depending upon thedosage form employed and the route of administration utilized.

In any of the aforementioned aspects are further embodiments comprisingsingle administrations of the effective amount of the pharmaceuticalcomposition of temsirolimus and a glucocorticoid described herein,including further embodiments in which the composition is administeredonce a month, twice a month, 3 times a month, once every 2 months, onceevery 3 months, once every 4 months, once every 5 months, or once every6 months.

In any of the aforementioned aspects are further embodiments comprisingsingle administrations of the effective amount of the pharmaceuticalcomposition of paclitaxel and a glucocorticoid described herein,including further embodiments in which the composition is administeredonce a month, twice a month, 3 times a month, once every 2 months, onceevery 3 months, once every 4 months, once every 5 months, or once every6 months.

In certain embodiments wherein a patient's status does improve, the doseof drug being administered is temporarily reduced or temporarilysuspended for a certain length of time (e.g., a “drug holiday”). Inspecific embodiments, the length of the drug holiday is between 1 monthand 5 years, including by way of example only, 2 months, 3 months, 4months, 5 months, 6 months, 7 months, 1 year, 2 years, 3 years, 4 years,5 years, or more than 5 years. The dose reduction during a drug holidayis, by way of example only, by 10%400%, including by way of example only10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, and 100%. In further or alternative embodiments, themethod comprises a drug holiday, wherein the administration of thecompound is temporarily suspended or the dose of the compound beingadministered is temporarily reduced; at the end of the drug holiday,dosing of the compound is resumed. In one embodiment, the length of thedrug holiday varies from 6 months to 2 years. In one embodiment, thelength of the drug holiday is 1 year. In one embodiment, the length ofthe drug holiday is 2 years. In one embodiment, the length of the drugholiday is 3 years.

Devices for Administration

Described herein are devices and methods for administration ofpharmaceutical compositions. As an alternative to stent-based luminaldrug delivery, the direct delivery of drug into vascular and otherluminal walls in some instances enhances the therapeutic concentrationsof pharmaceutical agents in targeted tissues. For example, it is in somecases particularly desirable to provide for an extended volumetricdistribution of the delivered pharmaceutical agent including bothlongitudinal and radial spreading from the injection site(s) in order toprovide therapeutic dosage levels of the agent within the targetedtissue region. In some instances, devices efficiently deliver the drugsinto the targeted tissue and limit or avoid the loss of drugs into theluminal blood flow. In some instances, the persistence of suchtherapeutic concentrations of the pharmaceutical agent in the tissuewere also increased, particularly in targeted tissues away from theblood vessel wall, including the adventitial tissue surrounding theblood vessel wall. In some instances, the uniformity and extent ofpharmaceutical agent delivery over remote, extended, and distributedregions of the adventitia and other tissues surrounding the bloodvessels is increased. In some instances, pharmaceutical agents aredelivered through the blood vessel walls at non-diseased sites withinthe blood vessel, where the agent then migrate through the adventitia orother tissues to the diseased site(s). In some instances, intravasculardelivery of pharmaceutical agents treats diseases and conditions of thetissues and organs in addition to those directly related to thevasculature.

In some cases, drug injection or infusion catheters and devices aresuitable for use with the methods described herein to injectpharmaceutical compositions into blood vessels the treat restenosis. Anexample of a device includes the Mercator Bullfrog® Micro-InfusionDevice available from Mercator MedSystems of Emeryville, CA. Otherexamples include the devices described in U.S. patent applications Ser.No. 14/605,865 and Ser. No. 15/691,138, the entire disclosures of whichare incorporated herein by reference. Examples of suitable devices andtheir use are described as follows. In some instances, injections areperformed using needles or microneedles.

In some cases, a pharmaceutical composition to treat the vasculardisease is delivered to the tissue surrounding a blood vessel using adrug injection or infusion catheter. In one example of a drug injectionor infusion catheter as shown in FIGS. 1A-2B, a microfabricatedintraluminal catheter 10 includes an actuator 12 having an actuator body12 a and central longitudinal axis 12 b. The actuator body more or lessforms a C-shaped outline having an opening or slit 12 d extendingsubstantially along its length. A microneedle 14 is located within theactuator body, as discussed in more detail below, when the actuator isin its unactuated condition (furled state) (FIG. 1B). The microneedle ismoved outside the actuator body when the actuator is operated to be inits actuated condition (unfurled state) (FIG. 2B). In some instances,the actuator is capped at its proximal end 12 e and distal end 12 f by alead end 16 and a tip end 18, respectively, of a therapeutic catheter20. The catheter tip end serves as a means of locating the actuatorinside a body lumen by use of a radio opaque coatings or markers. Thecatheter tip also forms a seal at the distal end 12 f of the actuator.The lead end of the catheter provides the necessary interconnects(fluidic, mechanical, electrical or optical) at the proximal end 12 e ofthe actuator.

Retaining rings 22 a and 22 b are located at the distal and proximalends, respectively, of the actuator. The catheter tip is joined to theretaining ring 22 a, while the catheter lead is joined to retaining ring22 b. The retaining rings are made of a thin, on the order of 10 to 100microns (μm), substantially flexible but relatively non-distensiblematerial, such as Parylene (types C, D or N), or a metal, for example,aluminum, stainless steel, gold, titanium or tungsten. The retainingrings form a flexible but relatively non-distensible substantially“C”—shaped structure at each end of the actuator. In some cases thecatheter is joined to the retaining rings by, for example, a butt-weld,an ultra-sonic weld, integral polymer encapsulation or an adhesive suchas an epoxy.

The actuator body further comprises a central, expandable section 24located between retaining rings 22 a and 22 b. The expandable section 24includes an interior open area 26 for rapid expansion when an activatingfluid is supplied to that area. The central section 24 is made of athin, semi-flexible but relatively non-distensible or flexible butrelatively non-distensible, expandable material, such as a polymer, forinstance, Parylene (types C, D or N), silicone, polyurethane orpolyimide. The central section 24, upon actuation, is expandablesomewhat like a balloon-device.

The central section is capable of withstanding pressures of up to about200 psi upon application of the activating fluid to the open area 26.The material from which the central section is made of is flexible butrelatively non-distensible or semi-flexible but relativelynon-distensible in that the central section returns substantially to itsoriginal configuration and orientation (the unactuated condition) whenthe activating fluid is removed from the open area 26. Thus, in thissense, the central section is very much unlike a balloon which has noinherently stable structure.

The open area 26 of the actuator is connected to a delivery conduit,tube or fluid pathway 28 that extends from the catheter's lead end tothe actuator's proximal end. The activating fluid is supplied to theopen area via the delivery tube. The delivery tube often is constructedof Teflon© or other inert plastics. The activating fluid often is asaline solution or a radio-opaque dye.

In some instances, the microneedle 14 is located approximately in themiddle of the central section 24. However, as discussed below, this isnot necessary, especially when multiple microneedles are used. Themicroneedle is affixed to an exterior surface 24 a of the centralsection. The microneedle is affixed to the surface 24 a by an adhesive,such as cyanoacrylate. Alternatively, the microneedle often is joined tothe surface 24 a by a metallic or polymer mesh-like structure 30 (SeeFIG. 4 ), which is itself affixed to the surface 24 a by an adhesive.The mesh-like structure often is-made of, for instance, steel or nylon.

The microneedle includes a sharp tip 14 a and a shaft 14 b. Themicroneedle tip can provide an insertion edge or point. The shaft 14 bcan be hollow and the tip can have an outlet port 14 c, permitting theinjection of a pharmaceutical or drug into a patient. The microneedle,however, does not need to be hollow, as it often is configured like aneural probe to accomplish other tasks.

As shown, the microneedle extends approximately perpendicularly fromsurface 24 a. Thus, as described, the microneedle will movesubstantially perpendicularly to an axis of a lumen into which has beeninserted, to allow direct puncture or breach of body lumen walls.

The microneedle further includes a pharmaceutical or drug supplyconduit, tube or fluid pathway 14 d which places the microneedle influid communication with the appropriate fluid interconnect at thecatheter lead end. This supply tube often is formed integrally with theshaft 14 b, or it often is formed as a separate piece that is laterjoined to the shaft by, for example, an adhesive such as an epoxy.

In some instances, the needle 14 is a 30-gauge, or smaller, steelneedle. Alternatively, the microneedle is microfabricated from polymers,other metals, metal alloys or semiconductor materials. The needle, forexample, is made of Parylene, silicon or glass. Microneedles and methodsof fabrication are described in U.S. application Ser. No. 09/877,653,filed Jun. 8, 2001, entitled “Microfabricated Surgical Device”, assignedto the assignee of the subject application, the entire disclosure ofwhich is incorporated herein by reference.

The catheter 20, in use, is inserted through an opening in the body(e.g. for bronchial or sinus treatment) or through a percutaneouspuncture site (e.g. for artery or venous treatment) and moved within apatient's body passageways 32, until a specific, targeted region 34 isreached (see FIG. 3 ). The targeted region 34 is the site of tissuedamage or more usually will be adjacent the sites typically being within100 mm or less to allow migration of the therapeutic or diagnosticagent. As is well known in catheter-based interventional procedures, thecatheter 20 follows a guide wire 36 that has previously been insertedinto the patient. Optionally, the catheter 20 also follows the path of apreviously-inserted guide catheter (not shown) that encompasses theguide wire.

During maneuvering of the catheter 20, well-known methods of fluoroscopyor magnetic resonance imaging (MRI) can be used to image the catheterand assist in positioning the actuator 12 and the microneedle 14 at thetarget region. As the catheter is guided inside the patient's body, themicroneedle remains unfurled or held inside the actuator body so that notrauma is caused to the body lumen walls.

After being positioned at the target region 34, movement of the catheteris terminated and the activating fluid is supplied to the open area 26of the actuator, causing the expandable section 24 to rapidly unfurl,moving the microneedle 14 in a substantially perpendicular direction,relative to the longitudinal central axis 12 b of the actuator body 12a, to puncture a body lumen wall 32 a. In some instances, it takes onlybetween approximately 100 milliseconds and five seconds for themicroneedle to move from its furled state to its unfurled state.

The ends of the actuator at the retaining rings 22 a and 22 b remainfixed to the catheter 20. Thus, they do not deform during actuation.Since the actuator begins as a furled structure, its so-called pregnantshape exists as an unstable buckling mode. This instability, uponactuation, in some cases produces a large-scale motion of themicroneedle approximately perpendicular to the central axis of theactuator body, causing a rapid puncture of the body lumen wall without alarge momentum transfer. As a result, a microscale opening is producedwith very minimal damage to the surrounding tissue. Also, since themomentum transfer is relatively small, only a negligible bias force isrequired to hold the catheter and actuator in place during actuation andpuncture.

The microneedle aperture, in fact, travels with such force that it canenter body lumen tissue 32 b as well as the adventitia, media, or intimasurrounding body lumens. Additionally, since the actuator is “parked” orstopped prior to actuation, more precise placement and control overpenetration of the body lumen wall are obtained.

After actuation of the microneedle and delivery of the agents to thetarget region via the microneedle, the activating fluid is exhaustedfrom the open area 26 of the actuator, causing the expandable section 24to return to its original, furled state. This also causes themicroneedle to be withdrawn from the body lumen wall. The microneedle,being withdrawn, is once again sheathed by the actuator.

Various microfabricated devices can be integrated into the needle,actuator and catheter for metering flows, capturing samples ofbiological tissue, and measuring pH. The device 10, for instance, couldinclude electrical sensors for measuring the flow through themicroneedle as well as the pH of the pharmaceutical being deployed. Thedevice 10 could also include an intravascular ultrasonic sensor (IVUS)for locating vessel walls, and fiber optics, as is well known in theart, for viewing the target region. For such complete systems, highintegrity electrical, mechanical and fluid connections are provided totransfer power, energy, and pharmaceuticals or biological agents withreliability.

By way of example, the microneedle has have an overall length of betweenabout 200 and 3,000 microns (μm). The interior cross-sectional dimensionof the shaft 14 b and supply tube 14 d often is on the order of 20 to250 um, while the tube's and shaft's exterior cross-sectional dimensionoften is between about 100 and 500 μm. The overall length of theactuator body often is between about 5 and 50 millimeters (mm), whilethe exterior and interior cross-sectional dimensions of the actuatorbody can be between about 0.4 and 4 mm, and 0.5 and 5 mm, respectively.The gap or slit through which the central section of the actuatorunfurls has in some instances a length of about 4-40 mm, and across-sectional dimension of about 50-500 μm. The diameter of thedelivery tube for the activating fluid often is about 100 μm. Thecatheter size often is between 1.5 and 15 French (Fr).

Variations of the present disclosure include a multiple-bucklingactuator with a single supply tube for the activating fluid. Themultiple-buckling actuator includes multiple needles that can beinserted into or through a lumen wall for providing injection atdifferent locations or times.

For instance, as shown in FIG. 4 , the actuator 120 includesmicroneedles 140 and 142 located at different points along a length orlongitudinal dimension of the central, expandable section 240. Theoperating pressure of the activating fluid is selected so that themicroneedles move at the same time. Alternatively, the pressure of theactivating fluid is selected so that the microneedle 140 moves beforethe microneedle 142.

Specifically, the microneedle 140 is located at a portion of theexpandable section 240 (lower activation pressure) that, for the sameactivating fluid pressure, will buckle outwardly before that portion ofthe expandable section (higher activation pressure) where themicroneedle 142 is located. Thus, for example, if the operating pressureof the activating fluid within the open area of the expandable section240 is two pounds per square inch (psi), the microneedle 140 will movebefore the microneedle 142. It is only when the operating pressure isincreased to four psi, for instance, that the microneedle 142 will move.Thus, this mode of operation provides staged buckling with themicroneedle 140 moving at time t₁, and pressure p₁, and the microneedle142 moving at time t₂ and p₂, with t₁, and p₁, being less than t₂ andp₂, respectively.

This sort of staged buckling can also be provided with differentpneumatic or hydraulic connections at different parts of the centralsection 240 in which each part includes an individual microneedle.

Also, as shown in FIG. 5 , an actuator 220 could be constructed suchthat its needles 222 and 224A move in different directions. As shown,upon actuation, the needles move at angle of approximately 90° to eachother to puncture different parts of a lumen wall. A needle 224B (asshown in phantom) could alternatively be arranged to move at angle ofabout 180° to the needle 224A.

Referring now to FIG. 6 , a needle injection catheter 310 constructed inaccordance with the principles of the present disclosure comprises acatheter body 312 having a distal end 314 and a proximal 316. Usually, aguide wire lumen 313 will be provided in a distal nose 352 of thecatheter, although over-the-wire and embodiments which do not requireguide wire placement will also be within the scope of the presentdisclosure. A two-port hub 320 is attached to the proximal end 316 ofthe catheter body 312 and includes a first port 322 for delivery of ahydraulic fluid, e.g., using a syringe 324, and a second port 326 fordelivering the pharmaceutical agent, e.g., using a syringe 328. Areciprocatable, deflectable needle 330 is mounted near the distal end ofthe catheter body 312 and is shown in its laterally advancedconfiguration in FIG. 6 .

Referring now to FIG. 7 , the proximal end 314 of the catheter body 312has a main lumen 336 which holds the needle 330, a reciprocatable piston338, and a hydraulic fluid delivery tube 340. The piston 338 is mountedto slide over a rail 342 and is fixedly attached to the needle 330.Thus, by delivering a pressurized hydraulic fluid through a lumen 341tube 340 into a bellows structure 344, the piston 338 often is advancedaxially toward the distal tip in order to cause the needle to passthrough a deflection path 350 formed in a catheter nose 352.

As can be seen in FIG. 8 , the catheter 310 often is positioned in acoronary blood vessel BV, over a guide wire GW in a conventional manner.Distal advancement of the piston 338 causes the needle 330 to advanceinto luminal tissue T adjacent to the catheter when it is present in theblood vessel. The therapeutic or diagnostic agents often are introducedthrough the port 326 using syringe 328 in order to introduce a plume Pof agent in the cardiac tissue, as illustrated in FIG. 8 . The plume Pwill be within or adjacent to the region of tissue damage as describedabove.

The needle 330 in some cases extends the entire length of the catheterbody 312 or, more usually, will extend only partially into thetherapeutic or diagnostic agents delivery lumen 337 in the tube 340. Aproximal end of the needle can form a sliding seal with the lumen 337 topermit pressurized delivery of the agent through the needle.

The needle 330 will be composed of an elastic material, typically anelastic or super elastic metal, typically being nitinol or other superelastic metal. Alternatively, the needle 330 could be formed from anon-elastically deformable or malleable metal which is shaped as itpasses through a deflection path. The use of non-elastically deformablemetals, however, is less preferred since such metals will generally notretain their straightened configuration after they pass through thedeflection path.

In some cases, the bellows structure 344 is made by depositing paryleneor another conformal polymer layer onto a mandrel and then dissolvingthe mandrel from within the polymer shell structure. Alternatively, thebellows 344 could be made from an elastomeric material to form a balloonstructure. In a still further alternative, a spring structure can beutilized in, on, or over the bellows in order to drive the bellows to aclosed position in the absence of pressurized hydraulic fluid therein.

After the therapeutic material is delivered through the needle 330, asshown in FIG. 8 , the needle is retracted and the catheter eitherrepositioned for further agent delivery or withdrawn. In someembodiments, the needle will be retracted simply by aspirating thehydraulic fluid from the bellows 344. In other embodiments, needleretraction is assisted by a return spring, e.g., locked between a distalface of the piston 338 and a proximal wall of the distal tip 352 (notshown) and/or by a pull wire attached to the piston and running throughlumen 341.

FIGS. 9A-9E illustrate an exemplary process for fabricating a dualmodulus balloon structure or anchored membrane structure in accordancewith the principles of the present disclosure. The first step of thefabrication process is seen in FIG. 9A, in which a low modulus “patch”,or membrane, material 400 is layered between removable (e.g.dissolvable) substrates 401 and 402. The substrate 401 covers one entireface of the patch 400, while the substrate 402 covers only a portion ofthe opposite face, leaving an exposed edge or border region about theperiphery.

In FIG. 9B, a layer of a “flexible but relatively non-distensible”material 403 is deposited onto one side of the sandwich structure fromFIG. 9A to provide a frame to which the low-modulus patch is attached.This material is, for example, parylene N, C, or D, though it can be oneof many other polymers or metals. When the flexible but relativelynon-distensible material is parylene and the patch material is asilicone or siloxane polymer, a chemomechanical bond is formed betweenthe layers, creating a strong and leak-free joint between the twomaterials. The joint formed between the two materials usually has a peelstrength or interfacial strength of at least 0.05 N/mm², typically atleast 0.1 N/mm², and often at least 0.2 N/mm².

In FIG. 9C, the “flexible but relatively non-distensible” frame oranchor material 403 has been trimmed or etched to expose the substratematerial 402 so that it can be removed. Materials 401 and 402 isdissolvable polymers that can be removed by one of many chemicalsolvents. In FIG. 9D, the materials 401 and 402 have been removed bydissolution, leaving materials 400 and 403 joined edge-to-edge to formthe low modulus, or elastomeric, patch 400 within a frame of generallyflexible but relatively non-distensible material 403.

As shown in FIG. 9E, when positive pressure +ΔP is applied to one side405 of the structure, the non-distensible frame 403 deforms onlyslightly, while the elastomeric patch 400 deforms much more. The lowmodulus material in some instances has a material modulus which isalways lower than that of the high modulus material and is typically inthe range from 0.1 to 1,000 MPa, more typically in the range from 1 to250 MPa. The high modulus material in some instances has a materialmodulus in the range from 1 to 50,000 MPa, more typically in the rangefrom 10 to 10,000 MPa. The material thicknesses often ranges in bothcases from approximately 1 micron to several millimeters, depending onthe ultimate size of the intended product. For the treatment of mostbody lumens, the thicknesses of both material layers 402 and 403 are inthe range from 10 microns to 2 mm.

Referring to FIGS. 10A-10D, the elastomeric patch of FIGS. 9A-9D isintegrated into the intraluminal catheter of FIG. 1-5 . In FIG. 10A-D,the progressive pressurization of such a structure is displayed in orderof increasing pressure. In FIG. 10A, the balloon is placed within a bodylumen L. The lumen wall W divides the lumen from periluminal tissue T,or adventitia A*, depending on the anatomy of the particular lumen. Thepressure is neutral, and the non-distensible structure forms a U-shapedinvoluted balloon 12 similar to that in FIG. 1 in which a needle 14 issheathed. While a needle is displayed in this diagram, other workingelements including cutting blades, laser or fiber optic tips,radiofrequency transmitters, or other structures could be substitutedfor the needle. For all such structures, however, the elastomeric patch400 will usually be disposed on the opposite side of the involutedballoon 12 from the needle 14.

Actuation of the balloon 12 occurs with positive pressurization. In FIG.10B, pressure (+ΔP₁) is added, which begins to deform the flexible butrelatively non-distensible structure, causing the balloon involution tobegin its reversal toward the lower energy state of a round pressurevessel. At higher pressure +ΔP₂ in FIG. 10C, the flexible but relativelynon-distensible balloon material has reached its rounded shape and theelastomeric patch has begun to stretch. Finally, in FIG. 10D at stillhigher pressure +ΔP₃, the elastomeric patch has stretched out toaccommodate the full lumen diameter, providing an opposing force to theneedle tip and sliding the needle through the lumen wall and into theadventitia. Typical dimensions for the body lumens contemplated in thisfigure are between 0.1 mm and 50 mm, more often between 0.5 mm and 20mm, and most often between 1 mm and 10 mm. The thickness of the tissuebetween the lumen and adventitia is typically between 0.001 mm and 5 mm,more often between 0.01 mm and 2 mm and most often between 0.05 mm and 1mm. The pressure +ΔP useful to cause actuation of the balloon istypically in the range from 0.1 atmospheres to 20 atmospheres, moretypically in the range from 0.5 to 20 atmospheres, and often in therange from 1 to 10 atmospheres.

As illustrated in FIGS. 11A-11C, the dual modulus structure formedherein provides for low-pressure (i.e., below pressures that may damagebody tissues) actuation of an intraluminal medical device to placeworking elements such as needles in contact with or through lumen walls.By inflation of a constant pressure, and the elastomeric material willconform to the lumen diameter to provide full apposition. Dual modulusballoon 12 is inflated to a pressure +ΔP₃ in three different lumendiameters in FIGS. 11A, 11B, and 11C. for the progressively largerinflation of patch 400 provides optimal apposition of the needle throughthe vessel wall regardless of diameter. Thus, a variable diameter systemis created in which the same catheter often is employed in lumensthroughout the body that are within a range of diameters. This is usefulbecause most medical products are limited to very tight constraints(typically within 0.5 mm) in which lumens may be used. A system asdescribed in this disclosure in some cases accommodates severalmillimeters of variability in the luminal diameters for which they areuseful.

FIGS. 12A-12F show schematics of an exemplary treating of vasculardisease in a subject. FIG. 12A shows a blood vessel 1210 in the lowerlimb that is affected by atherosclerosis or a plaque 1220 of lumen ofthe blood vessel. FIG. 12B shows the affected blood vessel 1210 after arevascularization procedure such as angioplasty or atherectomy toincrease the lumen diameter of the blood vessel. The target region ofthe tissue surrounding the affected blood vessel in some cases has had arevascularization procedure previously. FIG. 12C shows the delivery ofthe treatment catheter 10 into the target region through the vasculatureof the subject. FIG. 12D shows the expansion of the expandable element12 of the treatment catheter to puncture into the target tissue 1260surrounding the blood vessel with the needle 14 of the treatmentcatheter. The expandable element 12 often is also known as an actuator.FIG. 12E shows the delivery of the pharmaceutical composition comprisingtemsirolimus, dexamethasone, paclitaxel, or a combination thereof 1270into the target tissue surrounding the blood vessel 1260. FIG. 12F showsthe withdrawal of the treatment catheter 10 after the collapse of theexpandable element 12 and withdrawal of the needle 14 from the targettissue 1260 surrounding the blood vessel.

FIG. 13 shows a flow chart of a method 1300 of treating vascular diseasein a subject. In a step 1305, a subject suitable for treating a vasculardisease is identified. The vascular disease in some instances is anyvascular disease described above and herein. In exemplary embodiments,the vascular disease is post-angioplasty restenosis. In a step 1310, ablood vessel or blood vessels in the subject to target for treatmentoften is identified. In some instances, the blood vessel is any bloodvessel described above and herein, such as a femoral artery. In a step1315, a treatment catheter often is prepared with a pharmaceuticalcomposition comprising temsirolimus and dexamethasone, althoughtemsirolimus, dexamethasone, paclitaxel, contrast media or combinationsthereof may be used as the therapeutic agent of choice. Alternativepharmaceutical compositions often are used as well, and the treatmentcatheter often comprises any of the drug injection and infusion devicesdescribed herein and above. In a step 1320, the catheter often isadvanced through the vasculature of the subject to the target region(s),such as target region(s) in the blood vessel where plaque has beencompressed by angioplasty. In a step 1325, the catheter often ispositioned at or near the target region(s) of the blood vessel. In astep 1330, an expandable element of the catheter often is expanded topuncture the target region with a needle on the balloon. The expandableelement often is an expandable segment, an expandable section, or aballoon of the treatment catheter. The needle often is a microneedle. Ina step 1335, the needle of the treatment catheter often is positionedinto the tissue surrounding the blood vessel so that the aperture of theneedle often is positioned at the target tissue. In a step 1340, atherapeutic amount of the pharmaceutical composition comprisingtemsirolimus and dexamethasone (or other combinations of agents aspreviously described) often is injected into the target tissuesurrounding the blood vessel. The target tissue often is adventitialtissue, perivascular tissue, or connective tissue surrounding a bloodvessel. In a step 1345, the needle often is withdrawn from the tissueand the expandable element often is collapsed. In a step 1350, thetreatment catheter with the collapsed expandable element and the needleoften is removed from the vasculature of the subject.

Although the above steps show FIG. 12 and method 1300 of treating avascular disease in FIG. 13 in accordance with embodiments, a person ofordinary skill in the art will recognize many variations based on theteaching described herein. The steps in some cases, are completed in adifferent order. Steps often are added or deleted. Some of the steps insome instances comprise sub-steps. Many of the steps often are repeatedas often as beneficial to the treatment.

EXAMPLES In-Vitro Protocols and Methods

To assess the ability of a range of drug or drug combinations and theireffects on cell activation, pro-inflammatory cytokine production andcytotoxicity in PDGF-stimulated aortic smooth muscle cells in vitro wasexamined. A range of concentrations of the mTOR inhibitors sirolimus andtemsirolimus and the glucocorticoid dexamethasone were screened, inaddition to combinations thereof, in comparison to the mitotic inhibitorpaclitaxel.

Example 1: Metabolic Activity of VSMCs in the Presence of Select Drugs

Cultured human aortic VSMCs (4×10⁵ cells/mL) were grown in complete DMEMin 96 well plates (Corning) and stimulated with PDGF at 10 ng/mL(Peprotech) O/N. Utilizing established protocols for VSMC stimulationmediated by the growth factor, PDGF, drug was incubated at concentrationranges from 0, 1, 10, 100, 500 nanomolar (nM), in the presence orabsence of 50 nM dexamethasone, for 12 or 48 hours. Drug-specificeffects on cellular metabolic activity, pro-inflammatory cytokineproduction and cytotoxicity were examined. As a cytotoxic control fordisruption of metabolic activity, we used actinomycin D (100 ng/mL or398 nM). As a negative control, vehicle (5% (v/v) sterile DMSO insaline) was used. Following an 8 hour incubation in the presence of drugand stim (PDGF), MTT substrate was added and incubated for an additional4 hrs prior to measurement. Absorbance readings were taken using amicrotiter plate reader (Tecan). Data represent the average absorbancereadings. Error bars represent the standard deviation amongstreplicates. FIG. 14A shows the percent inhibition of metabolic activityand proliferation in the presence of sirolimus orsirolimus+dexamethasone. FIG. 14B shows the percent inhibition ofmetabolic activity and proliferation in the presence of temsirolimus ortemsirolimus+dexamethasone. FIG. 14C shows the percent inhibition ofmetabolic activity and proliferation in the presence of paclitaxel orpaclitaxel+dexamethasone. Data represent the average of 9 replicatewells per condition and error bars represent the standard deviation ofthe 9 replicates.

In the presence of PDGF, conversion of MTT substrate into colorimetricproduct should occur to a significantly higher degree, as compared tomedia controls. Indeed, in FIG. 14A VSMCs stimulated with 10 ng/mL PDGFshowed increased accumulation of metabolite compared to vehicle control(5% DMSO (v/v) in saline) and cytotoxic control (the transcriptioninhibitor actinomycin D at 100 ng/mL). Drug, specifically, temsirolimus,sirolimus and paclitaxel at higher concentrations had inhibitoryactivity in the MTT assay (FIGS. 14A-C).

In yet a further experiment (FIG. 19A), cultured human aortic VSMCs(4×10⁵ cells/mL) were grown in complete DMEM in 96 well plates andstimulated with PDGF at 10 ng/mL (Peprotech). Single drug titrations oftemsirolimus (TEM), paclitaxel (PAC) or dexamethasone (DEX) wereadministered (0, 10, 50, 500 nM), or fixed high-dose paclitaxel ortemsirolimus were administered in combination with dexamethasonetitrations (10, 50, 500 nM). After 8 h incubation, MTT substrate wasadded and incubated for an additional 4 hrs prior to measurement (T=12hours). Absorbance readings were taken using a microtiter plate reader(Tecan). Data represent the average absorbance readings. Error barsrepresent the standard deviation amongst replicates. Theanti-proliferative concentrations of temsirolimus, paclitaxel anddexamethasone were further analyzed in this study for their ability toabrogate PDGF-mediated proliferation at doses (dose range 1, 10, 50 500nM). FIG. 19A presents the percent of inhibition that was identified inthe presence of single or combination drugs. Low doses of all drugs,administered individually, had minimal inhibitory properties, ascompared to high dose of each drug. Addition of dexamethasone tohigh-dose paclitaxel or temsirolimus increased the anti-proliferativeeffect in each case.

Example 2: VSMC TNFα Cytokine Production in the Presence of Select Drugs

Cultured human aortic VSMCs (4×10⁵ cells/mL) were grown in complete DMEMin 96 well plates (Corning) and stimulated with PDGF at 10 ng/mL(Peprotech) O/N. As a negative control (0 nM Drug)), vehicle (5% (v/v)sterile DMSO in saline) was used. Following a 48 hour incubation in thepresence of drug and stimulant (PDGF), 50 uL of supernatant from eachwell was collected and ELISAs were performed to measure TNFα levels.Pro-inflammatory cytokine expression was examined under these conditionsby measuring TNFα levels in cell supernatant after 48 h by enzyme-linkedimmunoassay (ELISA; Peprotech #900-T16, #900-T25). Absorbance readingswere taken using a microtiter plate reader (Tecan). FIG. 15A shows TNFαproduction in the presence of increasing concentrations of sirolimus orsirolimus+50 nM dexamethasone. FIG. 15B shows TNFα production in thepresence of increasing concentrations of temsirolimus or temsirolimus+50nM dexamethasone. FIG. 15C shows TNFα production in the presence ofincreasing concentrations of paclitaxel or paclitaxel+50 nMdexamethasone. Data represent the average of 9 replicate wells percondition and error bars represent the standard deviation of the 9replicates. mTOR inhibitors decreased TNFα production at higher dosesand the presence of dexamethasone decreased TNFα production whencombined with mTOR inhibitors. Dexamethasone alone strongly decreasedTNFα production in a dose-dependent manner. Conversely, paclitaxelactivated pro-inflammatory cytokine expression, which was ameliorated bythe addition of 50 nM dexamethasone.

In yet a further experiment (FIG. 19E) cultured human aortic VSMCs(4×10⁵ cells/mL) were grown in complete DMEM in 96 well plates (Corning)and stimulated with PDGF at 10 ng/mL (Peprotech). Single drug titrationsof temsirolimus (TEM), paclitaxel (PAC) or dexamethasone (DEX) wereadministered (0, 10, 50, 500 nM), or fixed high-dose paclitaxel ortemsirolimus were administered in combination with dexamethasonetitrations (10, 50, 500 nM). Cells were incubated for 48 h and TNFαlevels were measured by ELISA (Peprotech). Absorbance readings weretaken using a microtiter plate reader (Tecan). Data represent theaverage absorbance readings. Error bars represent the standard deviationamongst triplicate wells of triplicate plates. In this experiment,paclitaxel induced a dose-dependent upregulation of the pro-inflammatorycytokines TNFα, while temsirolimus and dexamethasone, administeredindividually, induced a dose-dependent reduction in the same cytokine.Dexamethasone similarly demonstrated anti-inflammatory effects in thisstudy. Furthermore, at high dose (500 nM) paclitaxel, TNFα expressionlevels are highest; addition of dexamethasone at any dose (10, 50, 500nM) significantly reduced pro-inflammatory cytokine production ofpaclitaxel.

Example 3: VSMC IL6 Cytokine Production in the Presence of Select Drugs

Cultured human aortic VSMCs (4×10⁵ cells/mL) were grown in complete DMEMin 96 well plates (Corning) and stimulated with PDGF at 10 ng/mL(Peprotech) O/N. As a negative control (0 nM Drug), vehicle (5% (v/v)sterile DMSO in saline) was used. Following a 48 hour incubation in thepresence of drug and stimulant (PDGF), 50 uL of supernatant from eachwell was collected and ELISAs were performed to measure IL6 levels.Pro-inflammatory cytokine expression was examined under these conditionsby measuring IL6 levels in cell supernatant after 48 h by enzyme-linkedimmunoassay (ELISA; Peprotech #900-T16, #900-T25). Absorbance readingswere taken using a microtiter plate reader (Tecan). FIG. 16A shows IL6production in the presence of increasing concentrations of sirolimus orsirolimus+50 nM dexamethasone. FIG. 16B shows IL6 production in thepresence of increasing concentrations of temsirolimus or temsirolimus+50nM dexamethasone. FIG. 16C shows IL6 production in the presence ofincreasing concentrations of paclitaxel or paclitaxel+50 nMdexamethasone. Data represent the average of 9 replicate wells percondition and error bars represent the standard deviation of the 9replicates. With the exception of paclitaxel, the mTOR inhibitors andthe glucocorticoid inhibited IL6 production in a dose-dependent manner(FIGS. 16A-C).

The percent change in TNFα and IL6 levels compared to vehicle controllevels (set as baseline) were also analyzed and are presented in FIGS.15A-C and FIGS. 16A-C, respectively. The mTOR inhibitors each decreasedTNFα levels when present at 50-500 nM. Paclitaxel induced IL6 and TNFαproduction by SMCs at all doses tested. Moreover, TNFα levels increasedsteadily as paclitaxel concentrations increased with no significantchange in IL6 levels between 50 and 500 nM drug. Dexamethasoneameliorated paclitaxel-induced TNFα and IL6 production in VSMCs. Alsovery noteworthy was the unexpected finding that mTOR inhibitors alonedecreased IL6 production in a dose-dependent manner and similar todexamethasone. mTOR inhibitor+dexamethasone showed an enhanced abilityto inhibit IL6 production by VSMCs.

In yet a further experiment (FIG. 19D) cultured human aortic VSMCs(4×10⁵ cells/mL) were grown in complete DMEM in 96 well plates (Corning)and stimulated with PDGF at 10 ng/mL (Peprotech). Single drug titrationsof temsirolimus (TEM), paclitaxel (PAC) or dexamethasone (DEX) wereadministered (0, 10, 50, 500 nM), or fixed high-dose paclitaxel ortemsirolimus were administered in combination with dexamethasonetitrations (10, 50, 500 nM). Cells were incubated for 48 h and IL6levels were measured by ELISA (Peprotech). Absorbance readings weretaken using a microtiter plate reader (Tecan). Data represent theaverage absorbance readings. Error bars represent the standard deviationamongst triplicate wells of triplicate plates. In this experiment,paclitaxel induced a dose-dependent upregulation of the pro-inflammatorycytokines IL6, while temsirolimus and dexamethasone, administeredindividually, induced a dose-dependent reduction in the same cytokine.Dexamethasone similarly demonstrated anti-inflammatory effects in thisstudy. Furthermore, at high dose (500 nM) paclitaxel, IL6 expressionlevels are highest; addition of dexamethasone at any dose (10, 50, 500nM) significantly reduced pro-inflammatory cytokine production ofpaclitaxel.

Example 4: Apoptosis in Drug-Treated VSMCs

Cultured human aortic VSMCs (4×10⁵ cells/mL) were grown in complete DMEMin 96 well plates (Corning) and stimulated with PDGF at 10 ng/mL(Peprotech). Drug (sirolimus; temsirolimus; paclitaxel; dexamethasone)or vehicle control was incubated for 12 hours. At harvest, cells weregently lysed and a Caspase 3 ELISA was performed (Cell SignalingTechnologies, Cat #7190). Absorbance readings were taken using amicrotiter plate reader (Tecan). FIG. 17A shows Caspase 3 activation inthe presence of increasing concentrations of sirolimus or sirolimus+50nM dexamethasone. FIG. 17B shows Caspase 3 activation in the presence ofincreasing concentrations of temsirolimus or temsirolimus+50 nMdexamethasone. FIG. 17C shows Caspase 3 activation in the presence ofincreasing concentrations of paclitaxel or paclitaxel+50 nMdexamethasone. Data represent the average of 9 replicate wells percondition and error bars represent the standard deviation of the 9replicates. Baseline levels of VSMC apoptosis were obtained usingvehicle controls and values were set to zero. The percent change inapoptosis in the presence of drug was determined from baseline (FIG.17A-C). Apoptosis was observed to be induced in a dose-dependent mannerfor all mTOR inhibitors and paclitaxel, with paclitaxel inducing up to a173% average increase in apoptosis under the test conditions.Interestingly, dexamethasone did not induce significant apoptosis. Dataalso showed that low dose dexamethasone decreased the mTORinhibitor-induced effects.

In yet another experiment (FIG. 19B) cultured human aortic VSMCs (4×10⁵cells/mL) were grown in complete DMEM in 96 well plates (Corning) andstimulated with PDGF at 10 ng/mL (Peprotech). Single drug titrations oftemsirolimus (TEM), paclitaxel (PAC) or dexamethasone (DEX) wereadministered (0, 10, 50, 500 nM), or fixed high-dose paclitaxel ortemsirolimus were administered in combination with dexamethasonetitrations (10, 50, 500 nM). Cells were then incubated for 12 hours. Atharvest, plates were centrifuged at 800 rpm in a clinical centrifuge,and supernatant was carefully removed. Cells were then fixed andpermeabilized and assayed for cytoplasmic caspase 3 levels by ELISA,according to manufacturer instructions (Cell Signaling Technologies).Absorbance readings were taken using a microtiter plate reader (Tecan).Data represent the average absorbance readings. Error bars represent thestandard deviation amongst replicates. With this experiment, werevisited whether paclitaxel, temsirolimus or dexamethasone could inducecytotoxicity through an apoptotic mechanism at lower doses (1, 10, 50,500 nM) by measuring the amount of activated Caspase 3 enzyme presentunder various treatment conditions. FIG. 19B presents the amount ofapoptosis inhibited compared to vehicle control. Briefly, paclitaxel, atall doses tested, was shown to promote apoptosis (lack of inhibition),which was rescued by the addition of dexamethasone. The same rescueeffect of dexamethasone at increasing concentrations was also observedwhen used in conjunction with high dose temsirolimus.

Example 5: Necrotic LDH Measurements in Drug-Treated VSMCs

Cultured human aortic VSMCs (4×10⁵ cells/mL) were grown in complete DMEMin 96 well plates (Corning) and stimulated with PDGF at 10 ng/mL(Peprotech). Drug (sirolimus; temsirolimus; paclitaxel; dexamethasone)or vehicle control was incubated for 12 hours. At harvest, plates werecentrifuged at 800 rpm in a clinical centrifuge, and supernatant wascarefully removed and assayed for lysed cells by measuring the LDHreleased into the culture media by ELISA, according to manufacturerinstructions. Absorbance readings were taken using a microtiter platereader (Tecan). Temsirolimus, sirolimus, paclitaxel and dexamethasonewere also assessed for toxicity using the LDH release assay to measurenecrosis and cytolysis in PDGF-treated VSMCs (FIG. 18A-C). Data in FIG.18A represent the LDH release in the presence of increasingconcentrations of sirolimus or sirolimus+50 nM dexamethasone. FIG. 18Bshows LDH release in the presence of increasing concentrations oftemsirolimus or temsirolimus+50 nM dexamethasone. FIG. 18C shows LDHrelease in the presence of increasing concentrations of paclitaxel orpaclitaxel+50 nM dexamethasone. Data represent the average of 9replicate wells per condition and error bars represent the standarddeviation of the 9 replicates. A dose-dependent relationship wasobserved with mTOR inhibitors and paclitaxel. Dexamethasone did notinduce LDH release at any dose and suppressed LDH release compared tovehicle. A 50 nM concentration of dexamethasone also decreased mTOR andpaclitaxel-mediated LDH release in these studies.

In yet a further experiment (FIG. 19C), cultured human aortic VSMCs(4×10⁵ cells/mL) were grown in complete DMEM in 96 well plates (Corning)and stimulated with PDGF at 10 ng/mL (Peprotech). Single drug titrationsof temsirolimus (TEM), paclitaxel (PAC) or dexamethasone (DEX) wereadministered (0, 10, 50, 500 nM), or fixed high-dose paclitaxel ortemsirolimus were administered in combination with dexamethasonetitrations (10, 50, 500 nM). Treated cells were then incubated for 12hours. At harvest, plates were centrifuged at 800 rpm in a clinicalcentrifuge, and supernatant was carefully removed and assayed for LDH byELISA according to manufacturer instructions. Absorbance readings weretaken using a microtiter plate reader (Tecan). Data represent theaverage absorbance readings. Error bars represent the standard deviationamongst replicates. Toxicity of paclitaxel, through a necroticmechanism, has been shown in our previous studies. In this study weassessed lower concentrations of drug 1, 10, 50, 500 nM to see if wecould identify a drug range that was safer more well-tolerated by VSMCs.FIG. 19C presents data showing that all concentrations of paclitaxeltested demonstrated necrotic cytotoxicity. Additionally, drugcombinations containing paclitaxel similarly demonstrated necroticactivity, however the presence of dexamethasone rescued this activity.On the contrary, toxicity of a necrotic nature was not observed fordexamethasone at any concentration tested.

Example 6: Porcine Model of Femoral Vessel Injury

In a porcine model of femoral artery injury, a dose of temsirolimus,dexamethasone, or a combination thereof is administered directly intothe tissue around an injured artery through a catheter with a needle.Porcine vascular anatomy is similar to human anatomy, allowing the studyof medical equipment intended for use in humans. Porcine vascularpathology allows for the development of stenotic arteries for the studyof anti-stenotic or anti-restenotic therapies intended for use inhumans.

In twenty-four Yorkshire pigs, the femoral artery in each leg (hind leg)is injured by angioplasty overstretch and followed with eithertemsirolimus, dexamethasone, combination of both drugs, or controlsaline injection, for bilateral injury and injection. The angioplastyballoon is selected to be 40-60% larger than the reference diameter ofthe artery to be injured and is delivered by a catheter to the targetinjury site by carotid artery access. The angioplasty balloon isinflated to 10-20 atmosphere of pressure three times for 30 seconds eachinflation at the target injury site. After the balloon is removed, theMercator MedSystems Bullfrog® Micro-Infusion Device microneedle catheteris used to deliver either temsirolimus, dexamethasone, combination ofboth drugs, or control saline by injection into the adventitia andperivascular tissue around the injured artery at the center of eachtarget injury site. The injections are administered under and verifiedby fluoroscopy. The animals are monitored before, during, and after theprocedure, ensuring that all animals survive without adverse incidentsuntil sacrifice.

Temsirolimus preparation. The 25 mg/mL of Torisel® (temsirolimus) isdiluted to 10 mg/ml with the supplied diluent and further diluted to 1.0mg/mL in 0.9% sodium chloride solution. Then, the 1.0 mg/mL temsirolimusis diluted in a ratio of 4 parts temsirolimus to 2 parts contrastmedium, IsoVUE 370, and 4 parts 0.9% sodium chloride solution, for afinal temsirolimus concentration of 400 μg/ml. This temsirolimuspreparation is subsequently administered in temsirolimus-treated grouppigs. Similarly, a control solution is prepared by mixing 0.9% sodiumchloride solution at 4:1 ratio with a contrast medium, Isovue-370. Thiscontrol solution is administered in control group pigs.

Dexamethasone preparation. A 10 mg/mL solution of dexamethasone isdiluted to 5 mg/mL in 0.9% sodium chloride solution. Then, the 5 mg/mLdexamethasone is mixed at 4:1 ratio with a contrast medium, Isovue-370,for a final dexamethasone concentration of 4 mg/mL. This dexamethasonepreparation is subsequently administered in dexamethasone -treated grouppigs.

Temsirolimus/dexamethasone combination preparation. The 1.0 mg/mLtemsirolimus and the 10 mg/mL dexamethasone solutions are mixed at a2:2:1 ratio with a contrast medium, Isovue-370, for a final temsirolimusconcentration of 0.4 mg/mL, and a final dexamethasone concentration of4.0 mg/mL. This temsirolimus/dexamethasone preparation is subsequentlyadministered in temsirolimus/dexamethasone-treated group pigs.

Temsirolimus-treated group. Six pigs receive a single dose oftemsirolimus (1.5 mL of 400 μg/mL temsirolimus) in the tissue aroundeach 3-cm injured femoral artery segment, for a total of two doses peranimal. In each case, all temsirolimus treated animals undergoperivascular infusion into the femoral artery adventitia. Two pigs aresacrificed at each time point of 3 days, 7 days, and 28 dayspost-procedure, and each pig is analyzed for histopathology,pharmacokinetics, and safety evaluation.

Dexamethasone-treated group. Six pigs receive a single dose ofdexamethasone (1.5 mL of 4.0 mg/mL dexamethasone) in the tissue aroundeach injured femoral artery, for a total of two doses per animal. Ineach case, all dexamethasone treated animals undergo perivascularinfusion into the femoral artery adventitia. Two pigs are sacrificed ateach time point of 3 days, 7 days, and 28 days post-procedure, and eachpig is analyzed for histopathology, pharmacokinetics, and safetyevaluation.

Temsirolimus/Dexamethasone combination-treated group. Six pigs receive asingle dose of the temsirolimus/dexamethasone composition (1.5 ml of 400μg/mL temsirolimus/4.0 mg/mL dexamethasone) in the tissue around eachinjured femoral artery, for a total of two doses per animal. In eachcase, all temsirolimus treated animals undergo perivascular infusioninto the femoral artery adventitia. Two pigs are sacrificed at each timepoint of 3 days, 7 days, and 28 days post-procedure, and each pig isanalyzed for histopathology, pharmacokinetics, and safety evaluation.

Control group. Six pigs serve as control animals. Each pig receives 2injury sites in femoral arteries, for a total of 12 injury sites amongthe 6 pigs. Each injury site receives 1.5 ml of 0.9% sodium chloride(saline) diluted 4:1 ratio with contrast medium (Isovue-370). Two pigsare sacrificed at each time point of 3 days, 7 days, and 28 dayspost-procedure, and each pig is analyzed for histopathology,pharmacokinetics, and safety evaluation.

All treatment and control group animals will successfully receive theirrespective injection administered directly to the adventitia andperivascular tissues of the femoral arteries. All injection sites exceptthe two control sites will have complete or partial circumferential andlongitudinal coverage of the target site by the injection.

Safety Evaluation. Local and systemic toxicity is assessed by clinicalobservations and clinical pathology either during the survival durationor by analysis of tissues post mortem.

Outcomes. Cellular proliferation as measured by Ki-67 expression, BrdUexpression, histopathology, pharmacokinetics, and safety evaluation willbe compared for treatment and control groups to ascertain the effect ofcombination therapy on restenosis in a porcine model.

Example 8: Porcine Model of Femoral Vessel Injury with SequentialCombination Therapy

The general method of Example 7 is followed, with the modification thatthe researcher wants to study the effects of sequential treatment withboth drugs. The combination temsirolimus/dexamethasone-treatment groupis removed, the temsirolimus group receives a dexamethasone injection 24hours after treatment, and the dexamethasone group receives atemsirolimus injection 24 hours after treatment. Outcomes are evaluatedin a similar manner, and the effect of sequential treatment isascertained.

Example 9: Porcine Model of Femoral Vessel Injury with Paclitaxel

The general method of Example 7 is followed, with the modification thatthe researcher replaces temsirolimus with paclitaxel, in dosagesdescribed herein. Outcomes are evaluated in a similar manner.

Example 10: Porcine Model of Femoral Vessel Injury with SequentialPaclitaxel Combination Therapy

The general method of Example 8 is followed, with the modification thatthe researcher replaces temsirolimus with paclitaxel, in dosagesdescribed herein. Outcomes are evaluated in a similar manner, and theeffect of sequential treatment is ascertained.

Example 11: In-Vitro Cytoxicity Study

A study was performed to assess the ability of a range of drug or drugcombinations to induce cytotoxicity in PDGF-stimulated aortic smoothmuscle and endothelial cells in vitro. Cultured human aortic VSMCs(4×10⁵ cells/mL) were grown in complete DMEM in 96 well plates andstimulated with PDGF at 10 ng/mL (Peprotech). A range of concentrationsof drug (SIR, sirolimus; TEM, temsirolimus; PAC, paclitaxel; DEX,dexamethasone) and vehicle control or cytotoxic control (ACT D,actinomycin D) were screened (FIG. 20 ). Utilizing protocols for EC andVSMC stimulation mediated by the growth factor, PDGF, cells wereincubated in drug at concentration ranges from 0, 1, 10, 100 nM, 1, 10,100 μM for 12 hours prior to assessment for cellular activation andcytotoxicity. Prior to harvest, BrdU was added to each well for 6 hoursfor DNA incorporation. Absorbance readings were taken using a microtiterplate reader (Tecan). Data represent the average absorbance readings.Error bars represent the standard deviation amongst replicates. Cellularactivation was monitored by metabolic activity and cellularproliferation. Metabolic activity was measured using the MTT assay tomonitor enzyme-dependent metabolite production while proliferation wasmeasured using 5-bromo-2′-deoxyuridine (BrdU) ELISA to monitor DNAsynthesis.

In the presence of sufficient nutrients and growth factors such as PDGF,cells expressing PDGF receptors, such as VSMCs and ECs up-regulate theircellular metabolism to support growth and proliferation. VSMCsstimulated with 10 ng/mL PDGF showed increased accumulation ofmetabolite compared to media control and cytotoxic control (thetranscription inhibitor actinomycin D at 100 ng/mL). Vehicle control (5%DMSO (v/v) in saline) did not interfere with metabolic activity.Temsirolimus, sirolimus and paclitaxel at higher concentrations hadinhibitory activity in the MTT assay. Cellular proliferation in thepresence of PDGF showed dose-dependent inhibition of BrdU incorporationcompared to vehicle control, and concentrations of temsirolimus,sirolimus and paclitaxel were identified that approached the degree ofinhibition of actinomycin D.

Example 12: Porcine Study of Temsirolimus

Overview. The purpose of the first study was to assess thepharmacokinetic profile of temsirolimus administered directly to theadventitia and perivascular tissues of femoral arteries via the MercatorMedSystems Bullfrog® Micro-Infusion Device. Specific histopathology andhistomorphometry endpoints were used to determine efficacy of thetemsirolimus therapy.

Design

The study design included a total of eleven (11) animals; 8 of whichreceived angioplasty overstretch followed by single dose of test articlein bilateral superficial femoral arteries administered for a totalin-life phase of 1 hour (n=2), 3 days (n=2), 7 days (n=2) and 28 days(n=2) and three additional animals that served as control by undergoingangioplasty overstretch followed by infusion of saline and surviving for3 days (n=1), 7 days (n=1) and 28 days (n=1). A carotid artery accesswas utilized for all interventional procedures. In each case, alltemsirolimus-treated animals underwent perivascular infusion into thefemoral artery adventitia. Animals survived to 1 hour (n=2), 3 days(n=2), 7 days (n=2) and 28 days (n=2), for detailed histopathology andsafety evaluation. Following survival to the designated endpoint,animals underwent blood collection, then were euthanized and necropsiedfollowed by histological examination.

A prospective study of a single dose (357 μg in 1.5 ml) of adventitiallydelivered study drug (Torisel®) was conducted. All doses contained 50%contrast medium for distribution tracking. Each pig received bilateralinjections in the mid femoral region. 2 pigs each were sacrificed at 1hour, 3 days, 7 days and 28 days, for a total of 8 pigs. This was donein two phases: The first phase used to confirm the presence andconcentrations of temsirolimus and sirolimus and utilized 3 animals, oneeach at 1 hour, 3 days and 7 days. The second phase, after confirmationof drug concentrations in the first phase, included the remaining 5 pigs(1 each at 1 hour, 3 days and 7 days and 2 at 28 days). 3 pigs were usedas control in which treatment sites were subjected to overstretch injuryfollowed by infusion of saline via the Mercator Bullfrog® catheter andsacrificed at 3 days, 7 days or 28 days (1 pig at each time point).

The injury model was angioplasty overstretch 40-60%, at least 10 atm, 3inflations of 30 sec each and 30 sec flow. Adventitial delivery was madedirectly after overstretch injuries. Whole blood samples were takenfollowing each infusion at 5 minutes, 20 minutes, 1 hour, and then 24hours and upon sacrifice. Whole blood samples were analyzed forcirculating temsirolimus and sirolimus concentrations. Arteries wereperfused with Lactated Ringers Solution (LRS), extracted, and cut intoserial 5 mm sections with every other section fixed in formalin (notperfusion fixed) for immunohistochemical analysis and every othersection was frozen for LC/MS/MS analysis of both temsirolimus andsirolimus levels. Histopathology and histomorphometry endpoints wereused to determine efficacy of the rapamycin therapy.

Results. All animals successfully received overstretch injury to thesuperficial femoral arteries followed by administration of the testarticle or control. All animals in both the test and control groupssuccessfully received the test or control article through the MercatorMedSystems Bullfrog® Micro-Infusion device without any complications andsurvived to the designated endpoint. The survival period was devoid ofany test article/device or procedure related events. The concentrationof temsirolimus in the mixture (comprised of 50% temsirolimus and 50%Isovue-370) was 357 μg per 1.5 mL (238 μg/mL), which represents theconcentration that the treatment animals received. As all treatmentanimals received dosages into both femoral arteries, the total dosegiven to each treatment animal was 714 μg. The mean whole blood baselinetemsirolimus levels were below the limits of quantitation (0.200 ng/mlfor sirolimus and 0.500 ng/ml for temsirolimus) in all swine prior todosing. Following dosing, the mean temsirolimus levels were highest at 1hour after the first injection (32.1±11.0 ng/mL) and decreased to2.4±1.0 ng/mL within 24 hours. Concentrations continued to decrease toalmost non-detectable levels by the third day and by day 7 onwards, allblood analyzed for sirolimus and temsirolimus was found to be below thelimits of quantitation.

Similar trends were observed in the sirolimus concentration in the localvascular tissue, but presence of temsirolimus was much more persistentand measured in the tissue up to 28 days post-dosing. Clinical pathologyover the course of the survival duration revealed that there were notest device/drug-related effects in hematology. Changes observed inclinical chemistry were considered within expected ranges for biologicaland/or procedure-related variation. There was no evidence of localtoxicity to the treated vessels and no evidence of local vascularirritation upon Torisel® injection with the Mercator MedSystemsMicro-Infusion Device. Overall injection of Torisel® with the MercatorMedSystems Bullfrog® Micro-Infusion Device appeared safe in this model.The injection procedure produced no mural injury that could bedetectable microscopically. There was no or minor structural injuryascribable to the overstretch angioplasty procedure at 0, 3 and 7 days.There was some evidence of compressive injury in the form of single cellnecrosis in the media and/or media hypocellularity. There was noevidence of thrombosis or stenosis. At 28 days two treated vesselsshowed visible balloon overstretch injury with optimal secondaryhealing. Mural inflammation was absent or very minimal at all timeperiods and was associated with the slight mural compressive oroverstretch injury noted above.

The treated vessels were fully or nearly fully healed as early as Day 7,generally showing a normal wall and occasionally displaying minimal tomild perivascular or adventitial fibrosis and low severity nonspecificand localized mural inflammation considered to be of no pathologicalsignificance. There was complete or near complete re-endothelializationand no or minimal to mild and non-stenosing neointima formation. Ki67staining indicated cellular proliferation in the control vessel wallpeaking on Day 7. Quantitative analysis of Ki67 positive nuclei showed atreatment-related decrease in average proliferation values at Day 3, Day7 and Day 28. The decrease was substantial and consistent along thevessel length. Staining for smooth muscle actin (SMA) showed SMApositive cells in the adventitia on Day 3 that increased slightlythrough Day 7 and Day 28 and was primarily associated withmyofibroblasts and to a lesser degree with neovascularization. Thisvariation reflected adventitial healing response to vessel injury.

Conclusion. Following temsirolimus administration via the MercatorMedSystems Bullfrog® Micro-Infusion Device, the mean temsirolimus sodiumphosphate levels in whole blood were highest at 1 hour post-dose anddecreased at 24 hours post-dose. By 3 days post dose, whole bloodtemsirolimus concentration had fallen to almost non-detectable levels.At 7 and 28 days post-dose, whole blood temsirolimus levels were belowthe limit of quantitation. At the time of necropsy on study day 28,there were persistent detectable levels of temsirolimus present in thelocal vascular tissues in the treated swine.

Evaluation of tissues from eleven (11) swine administered either theTorisel® treatment or the control in the adventitia of the peripheralfemoral arteries with the Micro-Infusion Catheter (Mercator MedSystems)and euthanized at Day 0 (1 hour post treatment), Day 3, Day 7, and Day28 post treatment showed no adverse or toxicologically meaningfulchanges in the treated vessels. There was no or minimal to mildprocedural injury that was healed at the end of the study (Day 28) andproduced no adverse consequences on the patency or healing of treatedvessels. Cellular proliferation was increased on Day 3 in the vesselwall and adventitia and peaked on Day 7 decreasing slightly thereafter.Notably there was a moderate to marked decrease in proliferation indicesin Torisel®-treated vessels throughout the vessel wall at all timeperiods (Day 3, 7 and 28) compared to the respective controls. Thisdecrease was considered to be treatment-related.

The data from the current study indicate that infusion of Torisel®directly into the adventitia of femoral arteries of swine via theMercator MedSystems Bullfrog® Micro-Infusion device does not produceevidence of local or systemic toxicity assessed by clinical observationsand clinical pathology either during the survival duration or byhistopathology in the analysis of tissues post mortem.

Example 13: Porcine Study of Temsirolimus

Overview. The purpose of the second preclinical study was two-fold: (1)Confirm the safety of up to 52 mg Torisel® (temsirolimus) (10 mg perperipheral artery×4 arteries and 4 mg per coronary artery×3 arteries),and 5.2 mg Torisel® (1 mg per peripheral artery×4 arteries and 0.4 mgper coronary artery×3 arteries), as compared to saline; all agentscontained 20% contrast medium and were delivered to the perivasculartissues around the coronary and peripheral arteries, and (2) Determinepharmacokinetic profile of temsirolimus and sirolimus in tissue andblood samples after Torisel® (temsirolimus) was delivered to theadventitia and perivascular tissue around femoral and coronary arteriesin a porcine model of balloon overstretch artery stenosis.

Methods. Twelve animals were evaluated for tissue safety andpharmacokinetic blood and tissue profiles. The animals received vesselinjury induced via oversizing a balloon at ˜20-30% overstretch, thenreceiving subsequent Bullfrog® catheter delivery of 4 mg/mL Torisel® orsaline placebo control into the adventitia and perivascular tissuearound the coronary and femoral arteries. Blood samples forpharmacokinetic analysis were taken at 5 minutes, 30 minutes, 1 hour, 24hours, 72 hours, 7 Days, 28 Days, and termination. Animals wereterminated at 90 days. Gross examination of all treated tissues andsurrounding structures was performed. One coronary and one femoralartery and surrounding tissues were collected en bloc and flash frozenfrom each animal (note: one animal did not have a coronary vesselharvested) for pharmacokinetic analysis. Histological analysis wasperformed on all other collected arterial tissues.

Results

Endpoint 1: Overall Animal Health. All animals were generally healthyand gained weight for the duration of the study. No adverse treatmentrelated clinical observations were noted. All physical exams were normalthroughout the study duration.

Endpoint 2: Tissue Response to the Drug. There was no mortality orsignificant abnormalities noted in the treated arteries at necropsy.Histologically, all arteries were patent and there were no luminalthrombi or occlusions present in any of the sections examined in thetest groups. There were no histological remarkable differences in allgraded parameters in the treated vessels between the test groups and thecontrol group at either treatment location. There were thin neointimalformation, medial SMC loss, and medial fibrosis present in the treatedcoronary and femoral arteries in the control and test groups which mostlikely were caused by balloon overstretching injury in this animalmodel. The morphometric measured and calculated parameters werecomparable between the control and test groups. The treatment did notcreate any clinically significant findings in the low and high testgroups and therefore, did not raise a safety concern in this animalmodel. Taken together these findings suggest the safety of thetreatments used in this animal model at the 90-day time point.

Endpoint 3: Whole Blood Temsirolimus and Sirolimus Levels. The bloodlevels for all treated animals peaked at early time points, declinedover time to Day 7, and were below LLOQ at 28 days and at termination.In general, observed concentrations were proportional to dose.

Endpoint 4: Homogenized Vascular Tissue Temsirolimus and SirolimusLevels. The tissue levels for all animals were all below LLOQ.

Conclusion. All animals survived the treatment procedure to 90 days withno related deficiencies. There was no observed vascular toxicity ofTorisel® in comparison to saline as assessed with histopathologicalanalysis.

Example 14: Porcine Study of Temsirolimus and Dexamethasone inCombination

Overview: The purpose of this study was to evaluate the safety andpharmokinetic profile of Torisel® and dexamethasone when delivered in anexaggerated dose to peripheral vasculature in a porcine model of balloonoverstretch artery stenosis.

Methods: Four juvenile porcine subjects were used on this study toassess tissue safety and pharmacokinetic profile. The animals receivedfemoral artery injury induced via oversizing a balloon (˜20-30%overstretch). Three animals received subsequent treatment with theBullfrog® catheter of Torisel® (2 mg/mL) with dexamethasone (6 mg/mL)and contrast medium (20%) into the adventitia and perivascular tissuearound the peripheral arteries. The fourth animal received subsequenttreatment with the Bullfrog® catheter of saline and contrast medium(20%) into the adventitia and perivascular tissue around the peripheralarteries. Blood samples were taken at baseline, and post-implant 20minutes, 1 hour, 4 hours, 24 hours, 3 days, 7 days, and just prior totermination for pharmacokinetic assessment. One animal had an earlydeath at day 4 post-implant. The other three animals were survived for14 days. At day 14, blood was collected, animals were euthanized and anecropsy was performed.

Results

Endpoint 1: Overall animal health. Three of four animals survived forthe study duration and were generally healthy throughout. A fourthanimal was found deceased. All surviving animals showed a positiveweight gain over the course of the study. The deceased animal waslethargic on day 1. Lethargy continued until the animal was founddeceased on day 4. On day 2, blood samples were drawn for analysis,though no conclusive results were obtained. Fever and tachycardia werenoted on day 3 prior to the animal being found deceased on day 4. Noantemortem diagnosis was made. No significant findings in clinicalpathology were noted.

Endpoint 2: Tissue response to the device. Both test and control animalsexhibit none to moderate changes in the vessel wall. Perivascular andskeletal muscle changes also ranged from none to moderate in both testand control animals. Mineralization was not noted in control animaltissues examined. However, as mineralization was not previously seenwith a dosage of 4 mg/mL temsirolimus, it is considered likely due tothe high ethanol concentration in the solution at this dose.

Endpoint 3: Blood temsirolimus and dexamethasone levels (pK assessment).Temsirolimus levels in the blood for all test animals peaked at earlytime points, declined over time to 7 days and were below the lower limitof quantitation (LLOQ) at termination. The dexamethasone levels inplasma also peaked at early time points and were below LLOQ at the24-hour time point and after. At 14 days, dexamethasone tissue levelswere all <LLOQ, and low levels of temsirolimus were found in both testanimals.

Endpoint 4: Homogenized vascular tissue temsirolimus and dexamethasonelevels. Levels of temsirolimus (50.3 and 97.5 ng/g, equivalent to 48.8nM and 94.7 nM, respectively) were found in vascular tissue at term forthe two animals treated with the test solution; no temsirolimus wasfound in the control animal vessels. Dexamethasone levels were <LLOQ(10.0 ng/mL homogenate, equivalent to 100 ng/g in tissue, or 255 nM) invascular tissue at term for all animals.

Example 15: Rabbit Study of Temsirolimus and Dexamethasone inCombination

Overview. A rabbit study was performed to evaluate the tissue effect ofTorisel® (Tem) and dexamethasone (Dex) when delivered as combined oralone to peripheral vasculature in a Watanabe heritable hyperlipidemic(WHHL) rabbit model of balloon overstretch artery stenosis. Each animalwas subject to balloon angioplasty and treatment in each of the two (2)external iliac arteries. Four subjects were assigned to each of threegroups: Torisel®, dexamethasone, or combination. Three subjects wereassigned to each of two groups: control (vehicle delivery) or balloononly. Two New Zealand white rabbits were assigned to treatment withballoon only in one iliac and were naive to any treatment in the otheriliac.

Methods. Twenty animals were evaluated for tissue effect ofperi-vascular injection of test and control articles. Twenty-two animalsunderwent procedure on this study, as two animals died early and werereplaced. The animals received vessel injury induced via oversizing aballoon (˜20-30% overstretch). Animals were divided into the six groupswith varying balloon injury targets and various combinations of test andcontrol articles. The test and control articles were injected into theperi-vascular tissue using the Bullfrog® catheter. Animals wererecovered and survived for ˜28 days. At ˜28 days, animals weresacrificed, a gross pathology performed and tissue harvested forhistopathology analysis.

Results

Endpoint 1: Overall animal health (moribundityl. A total of 22 animalswere utilized on this study. Two animals either died or were euthanizedearly. The reason for death or euthanasia in these animals were likelydue to anesthesia and surgical intervention, but not test/controlarticle related. While there were some instances of inappetance, noanimals were noted to experience clinically significant weight loss.Many rabbits were noted as hyperglycemic and hyperlipidemic, howeverthis is consistent with what would be expected from the genetic model ofthe WHHL rabbits. No other significant health issues were identifiedthroughout the course of the study.

Endpoint 2: Tissue response to the device. The combination therapy(Tem+Dex) group showed less neointimal thickness (47.40±14.84 um) andarea (0.19±0.06 mm²) versus temsirolimus, dexamethasone, and vehiclegroup. The % HHF-35 positive intimal/medial area, as a marker ofvascular smooth muscle cell viability, was smaller with combinationtherapy or temsirolimus alone relative to dexamethasone alone or vehiclegroup. The number of BrdU and TUNEL positive cells including intima andmedia showed no statistical difference among these groups.

Twenty of 22 animals survived until 28-day term and 1 of the 20 animalswas euthanized per vet orders on day 28 due to acute collapse. For theearly death animals, death was related to anesthesia and surgicalintervention, not test/control article. All other animals had normalin-life experiences based on the animal model and procedures performed.No vascular toxicity was noted by histopathology for any sample.

The combination therapy (Tem+Dex) group showed less neointimal thickness(47.40±14.84 μm) and area (0.19±0.06 mm²) versus temsirolimus,dexamethasone, and vehicle group. The % HHF-35 positive intimal/medialarea, as a marker of vascular smooth muscle cell viability, was smallerwith combination therapy or temsirolimus alone relative to dexamethasonealone or vehicle group. The number of BrdU and TUNEL positive cellsincluding intima and media showed no statistical difference among thesegroups. However, the balloon-only group did not exhibit significantneointima or stenosis, and therefore none of the therapies showedspecific benefit over the balloon-only group. The balloon-only groupalso did not exhibit similar proliferative markers to any of the othergroups, suggesting a lack of proliferative injury induction or adifferent stage of disease in that group of rabbits.

Conclusion. The delivery of Torisel® and dexamethasone to a peripheralvasculature in a Watanabe heritable hyperlipidemic (WHHL) rabbit modelof balloon overstretch artery stenosis was found to be safe andeffective. Animals survived the study duration of ˜28 days withoutsignificant deficiencies related to the test article. Histopathologyevaluation of the various groups showed less neointimal thickness andarea in animals treated with Torisel and dexamethasone when compared tothe Torisel only, dexamethasone only, and vehicle groups.

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the present disclosure. It should beunderstood that various alternatives to the embodiments of the presentdisclosure described herein may be employed in practicing the presentdisclosure. It is intended that the following claims define the scope ofthe invention and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

What is claimed is:
 1. A method of treating a vascular disease in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising a therapeutically effective amount oftemsirolimus or a pharmaceutically acceptable salt thereof and atherapeutically effective amount of dexamethasone or a pharmaceuticallyacceptable salt thereof, wherein the composition is administered bydirect injection to a disease site.